U.S. patent number 8,758,792 [Application Number 13/746,516] was granted by the patent office on 2014-06-24 for bone matrix compositions and methods.
This patent grant is currently assigned to Warsaw Orthopedic, Inc.. The grantee listed for this patent is Warsaw Orthopedic, Inc.. Invention is credited to Keyvan Behnam, Nanette Forsyth, Guobao Wei.
United States Patent |
8,758,792 |
Behnam , et al. |
June 24, 2014 |
Bone matrix compositions and methods
Abstract
Osteoinductive compositions and implants having increased
biological activities, and methods for their production, are
provided. The biological activities that may be increased include,
but are not limited to, bone forming; bone healing; osteoinductive
activity, osteogenic activity, chondrogenic activity, wound healing
activity, neurogenic activity, contraction-inducing activity,
mitosis-inducing activity, differentiation-inducing activity,
chemotactic activity, angiogenic or vasculogenic activity, and
exocytosis or endocytosis-inducing activity. In one embodiment, a
method for producing an osteoinductive composition comprises
providing partially demineralized bone, treating the partially
demineralized bone to disrupt the collagen structure of the bone,
and optionally providing a tissue-derived extract and adding the
tissue-derived extract to the partially demineralized bone. In
another embodiment, an implantable osteoinductive and
osteoconductive composition comprises partially demineralized bone,
wherein the collagen structure of the bone has been disrupted, and,
optionally, a tissue-derived extract.
Inventors: |
Behnam; Keyvan (Red Bank,
NJ), Wei; Guobao (Milltown, NJ), Forsyth; Nanette
(Bayville, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Warsaw Orthopedic, Inc. |
Warsaw |
IN |
US |
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Assignee: |
Warsaw Orthopedic, Inc.
(Warsaw, IN)
|
Family
ID: |
40156936 |
Appl.
No.: |
13/746,516 |
Filed: |
January 22, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130136777 A1 |
May 30, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12140044 |
Jun 16, 2008 |
8357384 |
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60957614 |
Aug 23, 2007 |
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60948979 |
Jul 10, 2007 |
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60944411 |
Jun 15, 2007 |
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Current U.S.
Class: |
424/422; 424/549;
606/909; 427/2.26 |
Current CPC
Class: |
A61K
35/12 (20130101); A61K 35/00 (20130101); A61L
27/3608 (20130101); A61L 27/3683 (20130101); A61L
27/3604 (20130101); A61K 35/32 (20130101); A61L
27/54 (20130101); A61L 27/3834 (20130101); A61L
27/3695 (20130101); A61F 2310/00359 (20130101); A61L
2430/02 (20130101); A61F 2002/30059 (20130101); A61F
2/28 (20130101); A61L 2430/40 (20130101) |
Current International
Class: |
A61K
35/00 (20060101); A61K 35/32 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2005/084578 |
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Sep 2005 |
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WO |
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Primary Examiner: Kemmerer; Elizabeth C
Attorney, Agent or Firm: Sorell Lenna & Schmidt LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION(S)
This application is a continuation application of U.S. application
Ser. No. 12/140,044, now U.S. Pat. No. 8,357,384, filed on Jun. 16,
2008, which claims the benefit of U.S. Patent Application Ser. No.
60/944,411 filed Jun. 15, 2007; U.S. Patent Application Ser. No.
60/948,979 filed Jul. 10, 2007; and U.S. Patent Application Ser.
No. 60/957,614 filed Aug. 23, 2007, the contents of all the
aforementioned applications are incorporated by reference herein.
Claims
The invention claimed is:
1. An osteoinductive composition, the composition comprising: heat
and gaseous supercritical carbon dioxide treated surface
demineralized bone particles, the bone particles ranging from
approximately 1 mm to approximately 4 mm in their longest
dimension, and being approximately 10 to approximately 50%
demineralized; demineralized bone matrix; and a delivery
vehicle.
2. The osteoinductive composition of claim 1, wherein the surface
demineralized bone particles comprise surface demineralized
allograft bone particles.
3. The osteoinductive composition of claim 1, wherein the surface
demineralized bone particles comprise surface demineralized
xenograft bone particles.
4. The osteoinductive composition of claim 1, wherein the bone
particles are approximately 10% demineralized.
5. The osteoinductive composition of claim 1, wherein the partially
demineralized bone particles have a collagen structure and wherein
the collagen structure of the bone has been disrupted.
6. The osteoinductive composition of claim 1, wherein the partially
demineralized bone particles have been treated with atmospheric
supercritical carbon dioxide at a temperature between approximately
100.degree. C. and 250.degree. C.
7. The osteoinductive composition of claim 1, wherein the
osteoinductive composition comprises a slurry, putty, or gel.
8. The osteoinductive composition of claim 1, wherein the
demineralized bone matrix comprises demineralized bone fibers.
9. The composition of claim 8, wherein the demineralized bone
fibers have a median length to median thickness ratio of at least
about 10:1 and up to about 500:1, a median length of from about 2
mm to about 400 mm, a medium width of from about 2 mm to about 5
mm, and a median thickness of from about 0.02 to about 2 mm.
10. The composition of claim 8, wherein the demineralized bone
fibers comprise pressed demineralized bone fibers.
11. The osteoinductive composition of claim 1, wherein the delivery
vehicle is a carrier.
12. The osteoinductive composition of claim 11, wherein the carrier
is glycerol and wherein the osteoinductive composition is
moldable.
13. The osteoinductive composition of claim 11, wherein the carrier
is glycerol, and wherein the osteoinductive composition is
extrudable.
14. The osteoinductive composition of claim 1, wherein the delivery
vehicle is a covering.
15. The osteoinductive composition of claim 14, wherein the
covering is a mesh.
16. The osteoinductive composition of claim 14, wherein the
covering is tubular.
Description
BACKGROUND
Introduction
Mammalian bone tissue is known to contain one or more proteinaceous
materials, presumably active during growth and natural bone
healing, that can induce a developmental cascade of cellular events
resulting in endochondral bone formation. The active factors have
variously been referred to in the literature as bone morphogenetic
or morphogenic proteins (BMPs), bone inductive proteins, bone
growth or growth factors, osteogenic proteins, or osteoinductive
proteins. These active factors are collectively referred to herein
as osteoinductive factors.
It is well known that bone contains these osteoinductive factors.
These osteoinductive factors are present within the compound
structure of cortical bone and are present at very low
concentrations, e.g., 0.003%. Osteoinductive factors direct the
differentiation of pluripotential mesenchymal cells into
osteoprogenitor cells that form osteoblasts. Based upon the work of
Marshall Urist as shown in U.S. Pat. No. 4,294,753, issued Oct. 13,
1981, proper demineralization of cortical bone exposes the
osteoinductive factors, rendering it osteoinductive, as discussed
more fully below.
Overview of Bone Grafts
The rapid and effective repair of bone defects caused by injury,
disease, wounds, or surgery is a goal of orthopaedic surgery.
Toward this end, a number of compositions and materials have been
used or proposed for use in the repair of bone defects. The
biological, physical, and mechanical properties of the compositions
and materials are among the major factors influencing their
suitability and performance in various orthopaedic
applications.
Autologous cancellous bone ("ACB"), also known as autograft or
autogenous bone, long has been considered the gold standard for
bone grafts. ACB is osteoinductive and nonimmunogenic, and, by
definition, has all of the appropriate structural and functional
characteristics appropriate for the particular recipient.
Unfortunately, ACB is only available in a limited number of
circumstances. Some individuals lack ACB of appropriate dimensions
and quality for transplantation, and donor site pain and morbidity
can pose serious problems for patients and their physicians.
Bone grafting applications are differentiated by the requirements
of the skeletal site. Certain applications require a "structural
graft" in which one role of the graft is to provide mechanical or
structural support to the site. Such grafts contain a substantial
portion of mineralized bone tissue to provide the strength needed
for load-bearing. Examples of applications requiring a "structural
graft" include intercalary grafts, spinal fusion, joint plateaus,
joint fusions, large bone reconstructions, etc. Other applications
require an "osteogenic graft" in which one role of the graft is to
enhance or accelerate the growth of new bone tissue at the site.
Such grafts contain a substantial portion of demineralized bone
tissue to improve the osteoinductivity needed for growth of new
bone tissue. Examples of applications requiring "osteogenic graft"
include deficit filling, spinal fusions, joint fusions, etc. Grafts
may also have other beneficial biological properties, such as, for
example, serving as delivery vehicles for bioactive substances.
Bioactive substances include physiologically or pharmacologically
active substances that act locally or systemically in the host.
When mineralized bone is used in osteoimplants, it is primarily
because of its inherent strength, i.e., its load-bearing ability at
the recipient site. The biomechanical properties of osteoimplants
upon implantation are determined by many factors, including the
specific site from which the bone used to make the osteoimplant is
taken; various physical characteristics of the donor tissue; and
the method chosen to prepare, preserve, and store the bone prior to
implantation, as well as the type of loading to which the graft is
subjected.
Structural osteoimplants are conventionally made by processing, and
then machining or otherwise shaping cortical bones collected for
transplant purposes. Osteoimplants may comprise monolithic bone of
an aggregate of particles. Further, osteoimplants may be
substantially solid, flowable, or moldable. Cortical bone can be
configured into a wide variety of configurations depending on the
particular application for the structural osteoimplant. Structural
osteoimplants are often provided with intricate geometries, e.g.,
series of steps; concave or convex surfaces; tapered surfaces; flat
surfaces; surfaces for engaging corresponding surfaces of adjacent
bone, tools, or implants, hex shaped recesses, threaded holes;
serrations, etc.
One problem associated with many monolithic structural
osteoimplants, particularly those comprising cortical bone, is that
they are never fully incorporated by remodeling and replacement
with host tissue. Since repair is a cellular-mediated process, dead
(non-cellular, allograft or xenograft) bone is unable to repair
itself. When the graft is penetrated by host cells and host tissue
is formed, the graft is then capable of repair. It has been
observed that fatigue damage is usually the result of a buildup of
unrepaired damage in the tissue. Therefore, to the extent that the
implant is incorporated and replaced by living host bone tissue,
the body can then recognize and repair damage, thus eliminating
failure by fatigue. In applications where the mechanical
load-bearing requirements of the osteoimplant are challenging,
e.g., intervertebral spinal implants for spinal fusion, lack of
substantially complete replacement by host bone tissue may
compromise the osteoimplant by subjecting it to repeated loading
and cumulative unrepaired damage in the tissue (mechanical fatigue)
within the implant material. Thus, it is desirable that the
osteoimplant has the capacity to support load initially and be
capable of gradually transferring this load to the host bone tissue
as it remodels the implant.
Much effort has been invested in the identification and development
of alternative bone graft materials. Urist published seminal
articles on the theory of bone induction and a method for
decalcifying bone, i.e., making demineralized bone matrix (DBM).
Urist M. R., Bone Formation by Autoinduction, Science 1965;
150(698):893-9; Urist M. R. et al., The Bone Induction Principle,
Clin. Orthop. Rel. Res. 53:243-283, 1967. DBM is an osteoinductive
material in that it induces bone growth when implanted in an
ectopic site of a rodent, owing to the osteoinductive factors
contained within the DBM. It is now known that there are numerous
osteoinductive factors, e.g., BMP2, BMP4, BMP6, BMP7, which are
part of the transforming growth factor-beta (TGF-beta) superfamily.
BMP-2 has become the most important and widely studied of the BMP
family of proteins. There are also other proteins present in DBM
that are not osteoinductive alone but still contribute to bone
growth, including fibroblast growth factor-2 (FGF-2), insulin-like
growth factor-I and -II (IGF-I and IGF-II), platelet derived growth
factor (PDGF), and transforming growth factor-beta 1
(TGF-beta.1).
Accordingly, a known technique for promoting the process of
incorporation of osteoimplants is demineralization of portions of,
or the entire volume of, the implant. The process of demineralizing
bone grafts is well known. In this regard see, Lewandrowski et al.,
J. Biomed Materials Res, 31, pp. 365 372 (1996); Lewandrowski et
al., Calcified Tiss. Int., 61, pp. 294 297 (1997); Lewandrowski et
al., J. Ortho. Res., 15, pp. 748 756 (1997), the contents of each
of which is incorporated herein by reference.
DBM implants have been reported to be particularly useful (see, for
example, U.S. Pat. Nos. 4,394,370, 4,440,750, 4,485,097, 4,678,470,
and 4,743,259; Mulliken et al., Calcif Tissue Int. 33:71, 1981;
Neigel et al., Opthal. Plast. Reconstr. Surg. 12:108, 1996;
Whiteman et al., J. Hand. Surg. 18B:487, 1993; Xiaobo et al., Clin.
Orthop. 293:360, 1993, each of which is incorporated herein by
reference). DBM typically is derived from cadavers. The bone is
removed aseptically and treated to kill any infectious agents. The
bone is particulated by milling or grinding, and then the mineral
component is extracted by various methods, such as by soaking the
bone in an acidic solution. The remaining matrix is malleable and
can be further processed and/or formed and shaped for implantation
into a particular site in the recipient. The demineralized bone
particles or fibers can be formulated with biocompatible excipients
to enhance surgical handling properties and conformability to the
defect or surgery site. Demineralized bone prepared in this manner
contains a variety of components including proteins, glycoproteins,
growth factors, and proteoglycans. Following implantation, the
presence of DBM induces cellular recruitment to the site of injury.
The recruited cells may eventually differentiate into bone forming
cells. Such recruitment of cells leads to an increase in the rate
of wound healing and, therefore, to faster recovery for the
patient.
Demineralization provides the osteoimplant, whether monolithic,
aggregate, flowable, or moldable, with a degree of flexibility.
However, removal of the mineral components of bone significantly
reduces mechanical strength of bone tissue. See, Lewandrowski et
al., Clinical Ortho. Rel. Res., 317, pp. 254 262 (1995). Thus,
demineralization sacrifices some of the load-bearing capacity of
cortical bone and as such may not be suitable for all osteoimplant
designs.
While the collagen-based matrix of DBM is relatively stable, the
osteoinductive factors within the DBM matrix are rapidly degraded.
The osteogenic activity of the DBM may be significantly degraded
within 24 hours after implantation, and in some instances the
osteogenic activity may be inactivated within 6 hours. Therefore,
the osteoinductive factors associated with the DBM are only
available to recruit cells to the site of injury for a short time
after transplantation. For much of the healing process, which may
take weeks to months, the implanted material may provide little or
no assistance in recruiting cells. Further, most DBM formulations
are not load-bearing.
Extracting Proteins
The potential utility of osteoinductive factors has been recognized
widely. It has been contemplated that the availability of
osteoinductive factors could revolutionize orthopedic medicine and
certain types of plastic surgery, dental, and various periodontal
and craniofacial reconstructive procedures.
Urist's U.S. Pat. No. 4,294,753, herein incorporated by reference,
was the first of many patents on a process for extracting BMP from
DBM. At the time of the Urist '753 patent, BMP was referred to
generally. It is now known that there are multiple forms of BMP.
The Urist process became widely adopted, and though different users
may use different chemical agents from those disclosed in the basic
Urist process, the basic layout of the steps of the process remains
widely used today as one of the main methods of extracting BMP from
DBM. See, e.g., U.S. Pub 2003/0065392 (2003); U.S. Pub 2002/0197297
(2002). Urist reported that his basic process actually results in
generally low yields of BMP per unit weight of DBM.
Implanting Extracted Proteins
Successful implantation of the osteoinductive factors for
endochondral bone formation requires association of the proteins
with a suitable carrier material capable of maintaining the
proteins at an in vivo site of application. The carrier generally
is biocompatible, in vivo biodegradable, and sufficiently porous to
allow cell infiltration. Insoluble collagen particles that remain
after guanidine extraction and delipidation of pulverized bone
generally have been found effective in allogenic implants in some
species. However, studies have shown that while osteoinductive
proteins are useful cross species, the collagenous bone matrix
generally used for inducing endochondral bone formation is
species-specific. Sampath and Reddi, (1983) Proc. Nat. Acad. Sci.
USA 80: 6591-6594.
European Patent Application Serial No. 309,241, published Mar. 29,
1989, herein incorporated by reference, discloses a device for
inducing endochondral bone formation comprising an osteogenic
protein preparation, and a matrix carrier comprising 60-98% of
either mineral component or bone collagen powder and 2-40%
atelopeptide hypoimmunogenic collagen.
The use of pulverized exogenous bone growth material, e.g., derived
from demineralized allogenic or xenogenic bone, in the surgical
repair or reconstruction of defective or diseased bone in human or
other mammalian/vertebrate species is known. See, in this regard,
the disclosures of U.S. Pat. Nos. 4,394,370, 4,440,750, 4,472,840,
4,485,097, 4,678,470, 4,743,259, 5,284,655, 5,290,558; Bolander et
al., "The Use of Demineralized Bone Matrix in the Repair of
Segmental Defects," The Journal of Bone and Joint Surgery, Vol.
68-A, No. 8, pp. 1264-1273; Glowacki et al, "Demineralized Bone
Implants," Symposium on Horizons in Plastic Surgery, Vol. 12, No.
2; pp. 233-241 (1985); Gepstein et al., "Bridging Large Defects in
Bone by Demineralized Bone Matrix in the Form of a Powder," The
Journal of Bone and Joint Surgery, Vol. 69-A, No. 7, pp. 984-991
(1987); Mellonig, "Decalcified Freeze-Dried Bone Allograft as an
Implant Material In Human Periodontal Defects," The International
Journal of Periodontics and Restorative Dentistry, pp. 41-45 (June
1984); Kaban et al., "Treatment of Jaw Defects with Demineralized
Bone Implants," Journal of Oral and Maxillofacial Surgery, pp.
623-626 (Jun. 6, 1989); and Todescan et al., "A Small Animal Model
for Investigating Endosseous Dental Implants: Effect of Graft
Materials on Healing of Endosseous, Porous-Surfaced Implants Placed
in a Fresh Extraction Socket," The International Journal of Oral
& Maxillofacial Implants Vol. 2, No. 4, pp. 217-223 (1987), all
herein incorporated by reference.
A variety of approaches have been explored in an attempt to recruit
progenitor cells or chondrocytes into an osteochondral or chondral
defect. For example, penetration of subchondral bone has been
performed in order to access mesenchymal stem cells (MSCs) in the
bone marrow, which have the potential to differentiate into
cartilage and bone. Steadman, et al., "Microfracture: Surgical
Technique and Rehabilitation to Treat Chondral Defects," Clin.
Orthop., 391 S:362-369 (2001). In addition, some factors in the
body are believed to aid in the repair of cartilage. For example,
transforming growth factors beta (TGF-.beta.) have the capacity to
recruit progenitor cells into a chondral defect from the synovium
or elsewhere when loaded in the defect. Hunziker, et al., "Repair
of Partial Thickness Defects in Articular Cartilage: Cell
Recruitment From the Synovial Membrane," J Bone Joint Surg.,
78-A:721-733 (1996). However, the application of growth factors to
bone and cartilage implants has not resulted in the expected
increase in osteoinductive or chondrogenic activity.
U.S. Pat. No. 7,132,110, herein incorporated by reference,
describes an osteogenic composition prepared by a process including
the steps of subjecting demineralized bone to an extraction medium
to produce an insoluble extraction product and a soluble extraction
product, separating the insoluble extraction product and the
soluble extraction product, drying the soluble extraction product
to remove all or substantially all of the moisture in the soluble
extraction product, and combining the dried soluble extraction
product with demineralized bone particles. Studies using the
process have shown that the formed osteogenic composition does not
have appreciably increased osteoinductive properties when compared
to the demineralized bone particles to which the dried soluble
extraction product is added. It was further determined that the
demineralized bone from which the extraction products are extracted
does not exhibit appreciably decreased osteoinductive properties
when compared with its properties prior to extraction. It is thus
theorized that the extraction process withdraws only a small
fraction of available tissue repair factors.
Overall, current bone and cartilage graft formulations have various
drawbacks. The osteoinductive factors within the matrices can be
rapidly degraded and, thus, factors associated with the matrix are
only available to recruit cells to the site of injury for a short
time after implantation. Further, in certain instances the current
graft formulations exhibit limited capacity to stimulate tissue
formation.
BRIEF SUMMARY
Osteoinductive compositions and implants having increased
biological activities, and methods for their production, are
provided. The biological activities that may be increased include,
but are not limited to, bone forming, bone healing, osteoinductive
activity, osteogenic activity, chondrogenic activity, wound healing
activity, neurogenic activity, contraction-inducing activity,
mitosisinducing activity, differentiation-inducing activity,
chemotactic activity, angiogenic or vasculogenic activity, and
exocytosis or endocytosis-inducing activity.
In one embodiment, a method for producing an osteoinductive
composition is provided. The method comprises providing partially
demineralized bone, treating the partially demineralized bone to
disrupt the collagen structure of the bone, providing a
tissue-derived extract, and adding the tissue-derived extract to
the partially demineralized bone.
In another embodiment, an implantable osteoinductive and
osteoconductive composition is provided. The composition comprises
partially demineralized bone, wherein the collagen structure of the
bone has been disrupted, and a tissue-derived extract.
In yet another embodiment, a method for producing an osteoinductive
composition is provided. The method comprises providing surface
demineralized bone and treating the surface demineralized bone to
disrupt the collagen structure of the bone.
In a further embodiment, an implantable osteoinductive and
osteoconductive composition is provided. The composition comprises
surface demineralized bone or substantially fully demineralized,
wherein the collagen structure of the bone has been disrupted.
In yet a further embodiment, a method for treating a bone condition
is provided. The method comprises providing partially demineralized
bone, treating the partially demineralized bone to disrupt the
collagen structure of the bone, providing a tissue-derived extract,
adding the tissue-derived extract to the partially demineralized
bone, and implanting the tissue-derived extract and partially
demineralized bone.
In another embodiment, an osteoinductive composition is provided
comprising surface demineralized bone particles, the bone particles
ranging from approximately 1 mm to approximately 4 mm in length,
wherein the collagen structure of the bone has been disrupted. The
osteoinductive composition further comprises demineralized bone
matrix and tissue derived extract.
This application refers to various patents, patent applications,
journal articles, and other publications, all of which are
incorporated herein by reference. The following documents are
incorporated herein by reference: PCT/US04/43999; PCT/US05/003092;
US 2003/0143258 A1; PCT/US02/32941; Current Protocols in Molecular
Biology, Current Protocols in Immunology, Current Protocols in
Protein Science, and Current Protocols in Cell Biology, John Wiley
& Sons, N.Y., edition as of July 2002; Sambrook, Russell, and
Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Rodd 1989
"Chemistry of Carbon Compounds," vols. 1-5 and supps, Elsevier
Science Publishers, 1989; "Organic Reactions," vols 1-40, John
Wiley and Sons, New York, N.Y., 1991; March 2001, "Advanced Organic
Chemistry," 5th ed. John Wiley and Sons, New York, N.Y. In the
event of a conflict between the specification and any of the
incorporated references, the specification shall control. Where
numerical values herein are expressed as a range, endpoints are
included.
While multiple embodiments are disclosed, still other embodiments
of the present invention will become apparent to those skilled in
the art from the following detailed description. As will be
apparent, the invention is capable of modifications in various
obvious aspects, all without departing from the spirit and scope of
the present invention. Accordingly, the detailed description is to
be regarded as illustrative in nature and not restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a flowchart of a method for producing an
osteoinductive composition in accordance with one embodiment.
FIG. 2 illustrates a flowchart of a method for producing
osteoinductive bone in the absence of protease inhibitors in
accordance with one embodiment.
FIG. 3 illustrates a graph of neutral protease activity of
mineralized and demineralized bone.
FIG. 4a illustrates a generally round bone particle wherein the
bone particle has been surface demineralized in accordance with one
embodiment.
FIG. 4b illustrates an elongate bone particle wherein the bone
particle has been surface demineralized in accordance with one
embodiment.
FIG. 5 comparatively illustrates site response of autograft
implants versus site response of surface demineralized heat treated
particle implants.
DEFINITIONS
Bioactive Agent or Bioactive Compound, as used herein, to refers to
a compound or entity that alters, inhibits, activates, or otherwise
affects biological or chemical events. For example, bioactive
agents may include, but are not limited to, osteogenic or
chondrogenic proteins or peptides, anti-AIDS substances,
anti-cancer substances, antibiotics, immunosuppressants, anti-viral
substances, enzyme inhibitors, hormones, neurotoxins, opioids,
hypnotics, anti-histamines, lubricants, tranquilizers,
anti-convulsants, muscle relaxants and anti-Parkinson substances,
anti-spasmodics and muscle contractants including channel blockers,
miotics and anti-cholinergics, anti-glaucoma compounds,
anti-parasite and/or anti-protozoal compounds, modulators of
cell-extracellular matrix interactions including cell growth
inhibitors and antiadhesion molecules, vasodilating agents,
inhibitors of DNA, RNA or protein synthesis, anti-hypertensives,
analgesics, anti-pyretics, steroidal and non-steroidal
anti-inflammatory agents, anti-angiogenic factors, angiogenic
factors, anti-secretory factors, anticoagulants and/or
antithrombotic agents, local anesthetics, ophthalmics,
prostaglandins, anti-depressants, anti-psychotic substances,
anti-emetics, and imaging agents. In certain embodiments, the
bioactive agent is a drug. In some embodiments, the bioactive agent
is a growth factor, cytokine, extracellular matrix molecule or a
fragment or derivative thereof, for example, a cell attachment
sequence such as RGD. A more complete listing of bioactive agents
and specific drugs suitable for use in the present invention may be
found in "Pharmaceutical Substances: Syntheses, Patents,
Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical
Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals,
Drugs, and Biologicals", Edited by Susan Budavari et al., CRC
Press, 1996; and the United States Pharmacopeia-25/National
Formulary-20, published by the United States Pharmacopeial
Convention, Inc., Rockville Md., 2001, each of which is
incorporated herein by reference.
Biocompatible, as used herein, refers to materials that, upon
administration in vivo, do not induce undesirable long-term
effects.
Bone, as used herein, refers to bone that is cortical, cancellous
or cortico-cancellous of autogenous, allogenic, xenogenic, or
transgenic origin.
Bone Fibers, as used herein, refer to elongate bone particles
comprising threads or filaments having a median length to median
thickness ratio of at least about 10:1 and up to about 500:1, a
median length of from about 2 mm to about 400 mm, a medium width of
about 2 mm to about 5 mm, and a median thickness of from about 0.02
mm to about 2 mm.
Bone Particle, as used herein, refers to a piece of particulated
bone with wide range of average particle size ranging from
relatively fine powders to coarse grains and even larger chips. For
example, the bone particles may range in average particle size from
about 0.1 mm to about 15 mm in its largest dimension, or from about
0.5 to about 1.0 mm. The bone particles may be generally round and
have a radius, may be elongated, may be irregular, or may be in any
other suitable configuration. The bone particles can be obtained
from about cortical, cancellous and/or corticocancellous
autogenous, allogeneic, xenogeneic, or transgenic bone tissue.
Demineralized, as used herein, refers to any material generated by
removing mineral material from tissue, e.g., bone tissue. In
certain embodiments, the demineralized compositions described
herein include preparations containing less than 5% calcium. In
some embodiments, the demineralized compositions may comprise less
than 1% calcium by weight. Partially demineralized bone is intended
to refer to preparations with greater than 5% calcium by weight but
containing less than 100% of the original starting amount of
calcium. In some embodiments, demineralized bone has less than 95%
of its original mineral content. Percentage of demineralization may
refer to percentage demineralized by weight, or to percentage
demineralized by depth, as described with reference to FIGS. 4a and
4b. "Demineralized" is intended to encompass such expressions as
"substantially demineralized," "partially demineralized," "surface
demineralized," and "fully demineralized." "Partially
demineralized" is intended to encompass "surface
demineralized."
Demineralized bone matrix (DBM), as used herein, refers to any
material generated by removing mineral material from bone tissue.
In some embodiments, the DBM compositions as used herein include
preparations containing less than 5% calcium and preferably less
than 1% calcium by weight. In other embodiments, the DBM
compositions comprise partially demineralized bone (e.g.,
preparations with greater than 5% calcium by weight but containing
less than 100% of the original starting amount of calcium).
Osteoconductive, as used herein, refers to the ability of a
non-osteoinductive substance to serve as a suitable template or
substance along which bone may grow.
Osteogenic, as used herein, refers to materials containing living
cells capable of differentiation into bone tissue.
Osteoimplant as used herein refers to any bone-derived implant
prepared in accordance with the embodiments of this invention and
therefore is intended to include expressions such as bone membrane,
bone graft, etc.
Osteoinductive, as used herein, refers to the quality of being able
to recruit cells from the host that have the potential to stimulate
new bone formation. Any material that can induce the formation of
ectopic bone in the soft tissue of an animal is considered
osteoinductive. For example, most osteoinductive materials induce
bone formation in athymic rats when assayed according to the method
of Edwards et al., "Osteoinduction of Human Demineralized Bone:
Characterization in a Rat Model," Clinical Orthopaedics & Rel.
Res., 357:219-228, December 1998, incorporated herein by reference.
In other instances, osteoinduction is considered to occur through
cellular recruitment and induction of the recruited cells to an
osteogenic phenotype. Osteoinductivity score refers to a score
ranging from 0 to 4 as determined according to the method of
Edwards et al. (1998) or an equivalent calibrated test. In the
method of Edwards et al., a score of "0" represents no new bone
formation; "1" represents 1%-25% of implant involved in new bone
formation; "2" represents 26-50% of implant involved in new bone
formation; "3" represents 51%-75% of implant involved in new bone
formation; and "4" represents >75% of implant involved in new
bone formation. In most instances, the score is assessed 28 days
after implantation. However, the osteoinductivity score may be
obtained at earlier time points such as 7, 14, or 21 days following
implantation. In these instances it may be desirable to include a
normal DBM control such as DBM powder without a carrier, and if
possible, a positive control such as BMP. Occasionally
osteoinductivity may also be scored at later timepoints such as 40,
60, or even 100 days following implantation. Percentage of
osteoinductivity refers to an osteoinductivity score at a given
time point expressed as a percentage of activity, of a specified
reference score. Osteoinductivity may be assessed in an athymic rat
or in a human. Generally, as discussed herein, an osteoinductive
score is assessed based on osteoinductivity in an athymic rat.
Pressed bone fibers, as used herein, refer to bone fibers formed by
applying pressure to bone stock. The bone utilized as the starting,
or stock, material may range in size from relatively small pieces
of bone to bone of such dimensions as to be recognizable as to its
anatomical origin. The bone may be substantially fully
demineralized, surface demineralized, partially demineralized, or
nondemineralized. In general, the pieces or sections of whole bone
stock can range from about 1 to about 400 mm, from about 5 to about
100 mm, in median length, from about 0.5 to about 20 mm, or from
about 2 to about 10 mm, in median thickness and from about 1 to
about 20 mm, or from about 2 to about 10 mm, in median width.
Forming bone fibers by pressing results in intact bone fibers of
longer length than other methods of producing elongate bone fibers,
with the bone fibers retaining more of the native collagen
structure. The bone may be particulated via pressure applied to the
bone, as discussed in U.S. Pat. No. 7,323,193, herein incorporated
by reference.
Proteases, as used herein, refers to protein-cleaving enzymes that
cleave peptide bonds that link amino acids in protein molecules to
generate peptides and protein fragments. A large collection of
proteases and protease families has been identified. Some exemplary
proteases include serine proteases, aspartyl proteases, acid
proteases, alkaline proteases, metalloproteases, carboxypeptidase,
aminopeptidase, cysteine protease, collagenase, etc. An exemplary
family of proteases is the proprotein convertase family, which
includes furin. Dubois et al., American Journal of Pathology (2001)
158(1):305316. Members of the proprotein convertase family of
proteases are known to proteolytically process proTGFs and proBMPs
to their active mature forms. Dubois et al., American Journal of
Pathology (2001) 158(1):305-316; Cui et al., The Embo Journal
(1998) 17(16):4735-4743; Cui et al., Genes & Development (2001)
15:2797-2802, each incorporated by reference herein.
Protease inhibitors, as used herein, refers to chemical compounds
capable of inhibiting the enzymatic activity of protein cleaving
enzymes (i.e., proteases). The proteases inhibited by these
compounds include serine proteases, acid proteases,
metalloproteases, carboxypeptidase, aminopeptidase, cysteine
protease, etc. The protease inhibitor may act specifically to
inhibit only a specific protease or class of proteases, or it may
act more generally by inhibiting most if not all proteases.
Preferred protease inhibitors are protein or peptide based and are
commercially available from chemical companies such as
Aldrich-Sigma. Protein or peptide-based inhibitors which adhere to
the DBM (or calcium phosphate or ceramic carrier) may be preferred
because they remain associated with the matrix providing a
stabilizing effect for a longer period of time than freely
diffusible inhibitors. Examples of protease inhibitors include
aprotinin, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF),
amastatin-HCl, alpha1-antichymotrypsin, antithrombin III,
alpha1-antitrypsin, 4-aminophenylmethane sulfonyl-fluoride (APMSF),
arphamenine A, arphamenine B, E-64, bestatin, CA-074, CA-074-Me,
calpain inhibitor I, calpain inhibitor II, cathepsin inhibitor,
chymostatin, diisopropylfluorophosphate (DFP), dipeptidylpeptidase
IV inhibitor, diprotin A, E-64c, E-64d, E-64, ebelactone A,
ebelactone B, EGTA, elastatinal, foroxymithine, hirudin, leuhistin,
leupeptin, alpha2macroglobulin, phenylmethylsulfonyl fluo4de
(PMSF), pepstatin A, phebestin, 1,10phenanthroline, phosphoramidon,
chymostatin, benzamidine HCl, antipain, epsilon aminocaproic acid,
N-ethylmaleimide, trypsin inhibitor,
1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK),
1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin
inhibitor, and sodium EDTA.
Stabilizing agent, as used herein, refers to any chemical entity
that, when included in a composition comprising bone matrix and/or
a growth factor, enhances the osteoinductivity of the composition
as measured against a specified reference sample. In most cases,
the reference sample will not contain the stabilizing agent, but in
all other respects will be the same as the composition with
stabilizing agent. The stabilizing agent also generally has little
or no osteoinductivity of its own and works either by increasing
the half-life of one or more of the active entities within the
composition as compared with an otherwise identical composition
lacking the stabilizing agent, or by prolonging or delaying the
release of an active factor. In certain embodiments, the
stabilizing agent may act by providing a barrier between proteases
and sugar-degrading enzymes thereby protecting the osteoinductive
factors found in or on the matrix from degradation and/or release.
In other embodiments, the stabilizing agent may be a chemical
compound that inhibits the activity of proteases or sugar-degrading
enzymes. In some embodiments, the stabilizing agent retards the
access of enzymes known to release and solubilize the active
factors. Half-life may be determined by immunological or enzymatic
assay of a specific factor, either as attached to the matrix or
extracted there from. Alternatively, measurement of an increase in
osteoinductivity half-life, or measurement of the enhanced
appearance of products of the osteoinductive process (e.g., bone,
cartilage or osteogenic cells, products or indicators thereof) is a
useful indicator of stabilizing effects for an enhanced
osteoinductive matrix composition. The measurement of prolonged or
delayed appearance of a strong osteoinductive response will
generally be indicative of an increase in stability of a factor
coupled with a delayed unmasking of the factor activity.
Superficially demineralized, as used herein, refers to bone-derived
elements possessing at least about 90 weight percent of their
original inorganic mineral content, the expression "partially
demineralized" as used herein refers to bone-derived elements
possessing from about 8 to about 90 weight percent of their
original inorganic mineral content and the expression "fully
demineralized" as used herein refers to bone containing less than
8% of its original mineral context.
DETAILED DESCRIPTION
I. Introduction
Osteoinductive compositions and implants and methods for their
production are provided. In various embodiments, the osteoinductive
compositions may comprise one or more of partially demineralized
(including surface demineralized) bone particles treated to disrupt
the collagen structure, a tissue-derived material or extract, and a
carrier. In some embodiments, the partially demineralized bone
particles may not be treated to disrupt the collagen structure. In
some embodiments, demineralized bone matrix, such as demineralized
bone fibers, may be added to the treated partially demineralized
bone particles. The combination of DBM and partially demineralized
bone particles may then further include a tissue-derived extract
and/or a carrier. Those of ordinary skill will appreciate that a
variety of embodiments or versions of the invention are not
specifically discussed below but are nonetheless within the scope
of the present invention, as defined by the appended claims.
According to certain embodiments, partially demineralized bone
particles are exposed to a treatment or condition that increases at
least one biological activity of the partially demineralized bone
particles. A tissue-derived extract may be added to the partially
demineralized bone particles. Alternatively, or additionally, the
partially demineralized bone particles may be added to a carrier.
In some embodiments, the partially demineralized bone particles may
function as a carrier for the tissue-derived extract. In some
embodiments, the partially demineralized treated particles may be
used without addition of an extract or a carrier. In some
embodiments, the partially demineralized particles may not be
treated.
In some embodiments, a method of producing autolyzed,
antigen-extracted, allogeneic bone in the absence of protease
inhibitors is provided.
FIG. 1 illustrates a method 10 for producing an osteoinductive
composition in accordance with a first embodiment. As shown, the
method comprises particulating bone [block 12] and
surface-demineralizing the bone particles [block 14]. The surface
demineralized bone particles may be treated to disrupt collagen
structure of the bone [block 16]. The treatment may be done in any
suitable manner and is discussed more fully below. In some
embodiments, treatment of the surface demineralized bone particles
[block 16] is not done. A tissue-derived extract may added to the
surface-demineralized bone particles [block 18]. In some
embodiments, the surface-demineralized bone particles may be
combined with demineralized bone matrix, such as pressed
demineralized bone fibers [block 17]. The surface-demineralized
bone particles, with or without demineralized bone matrix or tissue
derived extract, may be used with a delivery vehicle [block 19]. In
one embodiment, the delivery vehicle may be a carrier and the
composition may be added to a carrier [block 20]. In another
embodiment the delivery vehicle may be a covering and the
composition, including the surface-demineralized bone particles,
pressed demineralized bone fibers, tissue derived extract, and/or
carrier, may be provided in a covering [block 22]. The composition,
including delivery vehicle in some embodiments, may be used to
treat a bone defect [block 24].
In some embodiments, treatment of the surface of demineralized bone
particles [block 16] may disrupt collagen and growth factors of
both the exterior and the interior of the bone particles. In other
embodiments, collagen and growth factors of the exterior of the
bone may be left substantially intact while collagen and growth
factors of the interior of the bone are disrupted.
Surface demineralization of the bone substantially removes mineral
and proteases from the surface of the bone. FIG. 3 is a graph
showing neutral protease activity of mineralized and demineralized
bone. As shown, demineralized bone has significantly lower neutral
protease activity than mineralized bone. Demineralization prior to
autolysis or treatment of the bone reduces protease activity on the
surfaces of the particle. Accordingly, using treatment techniques
that disrupt collagen and growth factors in the presence of
proteases, for example, autolysis, surface collagen and growth
factors are not disrupted if demineralization proceeds such
treatment. In contrast, the growth factors in the mineralized
portion of the bone are disrupted during such treatment. The lower
protease activity of the particle surfaces maintains osteoinductive
activity. Autolysis of the osteoconductive mineralized core of the
particles causes the particles to exhibit reduced delayed
hypersensitivity reaction. Thus, in accordance with some
embodiments, a method of autolysis of bone and maintenance of
osteoinductive activity in the bone without requiring use of
protease inhibitors.
FIG. 2 illustrates a method 30 of producing osteoinductive bone in
the absence of protease inhibitors. As shown in FIG. 2, bone
particles are particulated [block 32]. The bone particles may be
particulated to any suitable size ranging from microns to
millimeters. In some embodiments, the particles are particulated to
a size ranging from approximately 500 microns to approximately 10
mm, from approximately 500 microns to approximately 4 mm, or other
size. In one embodiment, the bone particles range from between
about 0.5 mm to about 15 mm in their longest dimension. The bone
particles are delipidized [block 34]. Delipidizing the bones may
comprise delipidizing the bone in 70% to 100% ethanol for more than
about 1 hour. Delipidizing the bones may also comprise delipidizing
bone in a critical or supercritical fluid such as carbon dioxide.
The delipidized bone particles are surface demineralized [block
36], as described more fully below. The surface demineralized
delipidized bone particles may optionally be treated to disrupt
collagen by, for example, incubating in a phosphate buffer [block
38]. The incubation may be done in any suitable manner, including,
for example, at a pH of approximately 7.4, at approximately
37.degree. C. for several hours (for example, ranging from
approximately 2 hours to approximately 96 hours). The particles may
be treated to remove water, for example via lyophilization or
critical point drying [block 37], and sterilized [block 39]. In
some embodiments, removing water the particles may be done prior to
treating the surface demineralized bone particles to disrupt the
collagen structure. Removing water from the particles may be
referred to as drying the particles or dehydrating the particles
and may be done to any suitable level. Sterilization may comprise,
for example, treatment with supercritical carbon dioxide. The bone
particles may be used with a delivery vehicle [block 40], such as
by adding to a carrier [block 41] and/or placement in a covering
[block 42].
In some embodiments, demineralized bone fibers may be combined with
the bone particles in a delivery vehicle [block 43]. In some
embodiments, the bone fibers are formed by pressing, described
below. Prior to combination with the particles, water may be
removed from the bone fibers [block 45]. Drying of the pressed
fibers may comprise, for example, critical point drying. U.S. Pat.
No. 7,323,193 for a Method of Making Demineralized Bone Particles,
herein incorporated by reference, describes suitable methods for
making pressed demineralized bone fibers that may be used with the
present invention.
The bone particles provided by the methods of FIG. 1 or 2 may be
combined with tissue-derived extracts and/or carriers. In certain
embodiments, the tissue-derived extract includes collagen type-I or
collagen type-I residues. Thus, the extract may contain peptides or
protein fragments that increase the osteoinductive or chondrogenic
properties of the partially demineralized bone particles. Bone is
made up principally of cells, and also of collagen, minerals, and
other noncollagenous proteins. Bone matrices can be
nondemineralized, partially demineralized, demineralized,
deorganified, anorganic, or mixtures of these. DBM is comprised
principally of proteins and glycoproteins, collagen being the
primary protein component of DBM. While collagen is relatively
stable, normally being degraded only by the relatively rare
collagenase enzymes, various other proteins and active factors
present in DBM are quickly degraded by enzymes present in the host.
These host-derived enzymes include proteases and sugar-degrading
enzymes (e.g., endo- and exoglycosidases, glycanases, glycolases,
amylase, pectinases, galacatosidases, etc.). Many of the active
growth factors responsible for the osteoinductive activity of DBM
exist in cryptic form, in the matrix until activated. Activation
can involve the change of a pre or pro function of the factor,
release of the function from a second factor or entity that binds
to the first growth factor, or exposing the BMPs to make them
available at the outer surface of the DBM. Thus, growth factor
proteins in a DBM or added to a DBM may have a limited
osteoinductive effect because they are rapidly inactivated by the
proteolytic environment of the implant site, or even within the DBM
itself.
A number of endogenous factors that play important roles in the
development and/or repair of bone and/or cartilage have been
identified. BMPs such as BMP-2 and BMP-4 induce differentiation of
mesenchymal cells towards cells of the osteoblastic lineage,
thereby increasing the pool of mature cells, and also enhance the
functions characteristic of differentiated osteoblasts. Canalis et
al., Endocrine Rev. 24(2):218-235, 2003, herein incorporated by
reference. In addition, BMPs induce endochondral ossification and
chondrogenesis. BMPs act by binding to specific receptors, which
results in phosphorylation of a class of proteins referred to as
SMADs. Activated SMADs enter the nucleus, where they regulate
transcription of particular target genes. BMPs also activate
SMAD-independent pathways such as those involving Ras/MAPK
signaling. Unlike most BMPs such as BMP-2 and BMP-4, certain BMPs
(e.g., BMP-3) act as negative regulators (inhibitors) of
osteogenesis. In addition, BMP-1 is distinct both structurally and
in terms of its mechanism of action from other BMPs, which are
members of the TGF-.beta. superfamily. Unlike certain other BMPs
(e.g., BMP-2, BMP-4), BMP-1 is not osteoinductive. Instead, BMP-1
is a collagenolytic protein that has also been shown to cleave
chordin (an endogenous inhibitor of BMP-2 and BMP-4). Tolloid is a
metalloprotease that is structurally related to BMP-1 and has
proteolytic activity towards chordin. See Canalis, supra, for
further details regarding the activities of BMPs and their roles in
osteogenesis and chondrogenesis.
A variety of endogenous inhibitors of BMPs have been discovered in
addition to chordin. These proteins act as BMP antagonists and
include pseudoreceptors (e.g., Bambi) that compete with signaling
receptors, inhibitory SMADs that block signaling, intracellular
binding proteins that bind to activating SMADs, factors that induce
ubiquitination and proteolysis of activating SMADs, and
extracellular proteins that bind BMPs and prevent their binding to
signaling receptors. Among the extracellular proteins are noggin,
chordin, follistatin, members of the Dan/Cerberus family, and
twisted gastrulation.
II. Implantable Osteoinductive/Osteoconductive Composition
An implantable osteoinductive composition and methods for preparing
such composition are provided. The osteoinductive composition has
an increased biological activity compared to other demineralized
bone. For example, the composition may have inductivity exceeding
that of from greater than one to about two to about five equivalent
volumes of demineralized bone prepared by traditional, prior art
methods. The osteoinductive composition may be formed into an
implant and/or may be provided in a delivery vehicle.
The biological activities of the composition that may be increased
include but are not limited to osteoinductive activity, osteogenic
activity, chondrogenic activity, wound healing activity, neurogenic
activity, contraction-inducing activity, mitosis-inducing activity,
differentiation-inducing activity, chemotactic activity, angiogenic
or vasculogenic activity, and exocytosis or endocytosis-inducing
activity. It will be appreciated that bone formation processes
frequently include a first stage of cartilage formation that
creates the basic shape of the bone, which then becomes mineralized
(endochondral bone formation). Thus, in many instances,
chondrogenesis may be considered an early stage of osteogenesis,
though of course it may also occur in other contexts.
The osteoinductive composition may comprise all or some of
partially demineralized bone particles, demineralized bone fibers,
a tissue-derived extract, and a delivery vehicle. The
osteoinductive composition provides concentrated or enhanced
osteoinductive activity. In some embodiments, the osteoinductive
composition is prepared by providing partially demineralized bone,
optionally treating the partially demineralized bone, extracting
osteoinductive factors from tissue, and adding the extracted
osteoinductive factors to the partially demineralized bone. The
partially demineralized bone and extract may be added to a delivery
vehicle such as a carrier or a covering. In other embodiments, the
osteoinductive composition is prepared by provided partially
demineralized bone particles (which may be in the form of chips),
providing pressed demineralized bone fibers, and combining the
partially demineralized bone particles and pressed demineralized
bone fibers, for example in a delivery vehicle. The partially
demineralized bone, pressed demineralized bone fibers, extract, and
delivery vehicle may form an osteoimplant. The osteoimplant, when
implanted in a mammalian body, can induce at the locus of the
implant the full developmental cascade of endochondral bone
formation including vascularization, mineralization, and bone
marrow differentiation. Also, in some embodiments, the
osteoinductive composition can be used as a delivery device to
administer bioactive agents.
In some embodiments, the partially demineralized bone may comprise
the delivery vehicle by forming a carrier. In certain embodiments,
the carrier contains peptides or protein fragments that increase
its osteoinductive or chondrogenic properties. In some embodiments,
the carrier comprises the remaining matrix after extraction. The
tissue-derived extract, for example, peptides or protein fragments,
may be exogenously added to the carrier. Further, other agents may
be added to the carrier and/or to the partially demineralized bone,
e.g., agents that improve the osteogenic and/or chondrogenic
activity of the partially demineralized bone by either
transcriptional or post-transcriptional regulation of the synthesis
of bone or cartilage enhancing or inhibiting factors by cells
within the carrier.
III. Provide Partially Demineralized Bone
In some embodiments, demineralized bone that is substantially fully
demineralized is used. In other embodiments, partially
demineralized bone is used. In other embodiments, the surface
demineralized bone is used. In other embodiments, nondemineralized
bone may be used. In other embodiments, combinations of some of all
of the above may be used. While many of the examples in this
section refer to partially or surface demineralized bone, this is
for illustrative purposes.
In one embodiment, the bone is partially demineralized. Referring
to FIG. 1, the bone may be surface demineralized [block 14]. The
partially demineralized bone may be provided in any suitable
manner. Generally, the bone may be obtained utilizing methods well
known in the art, e.g., allogenic donor bone. The partially
demineralized bone may comprise monolithic bone, bone particles, or
other bone-derived elements. In some embodiments, the partially
demineralized bone comprises partially demineralized bone
particles. The particles may range in size from about 0.5 mm to
about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to
about 8 mm, from about 1 mm to about 4 mm, from about 0.5 mm to
about 4 mm, or other range, in their longest dimension.
Bone-derived elements can be readily obtained from donor bone by
various suitable methods, e.g., as described in U.S. Pat. No.
6,616,698, incorporated herein by reference. The bone may be
cortical, cancellous, or cortico-cancellous of autogenous,
allogenic, xenogenic, or transgenic origin. The demineralized bone
is referred to as partially demineralized for the purposes of
illustration. Partially demineralized bone as used herein includes
surface demineralized bone.
As will be described, the bone may be particulated, demineralized,
and treated.
Demineralized bone matrix (DBM) preparations have been used for
many years in orthopedic medicine to promote the formation of bone.
For example, DBM has found use in the repair of fractures, in the
fusion of vertebrae, in joint replacement surgery, and in treating
bone destruction due to underlying disease such as rheumatoid
arthritis. DBM is thought to promote bone formation in vivo by
osteoconductive and osteoinductive processes. The osteoinductive
effect of implanted DBM compositions is thought to result from the
presence of active growth factors present on the isolated
collagen-based matrix.
To provide the osteoinductive composition described herein, the
bone is treated to remove mineral from the bone. Generally, the
bone is partially or surface demineralized. While hydrochloric acid
is the industry-recognized demineralization agent of choice, the
literature contains numerous reports of methods for preparing DBM
(see, for example, Russell et al., Orthopaedics 22(5):524-53 1, May
1999; incorporated herein by reference). The partially
demineralized bone may be prepared by methods known in the art or
by other methods that can be developed by those of ordinary skill
in the art without undue experimentation. In some instances, large
fragments or even whole or monolithic bone may be demineralized.
The whole or monolithic bone may be used intact or may be
particulated following demineralization. In other embodiments, the
bone may be particulated and then demineralized, as shown in FIG.
1.
Any suitable demineralization procedure may be used. In one
demineralization procedure, the bone is subjected to an acid
demineralization step followed by a defatting/disinfecting step.
The bone is immersed in acid over time to effect demineralization.
Acids that can be employed in this step include inorganic acids
such as hydrochloric acid and as well as organic acids such as
formic acid, acetic acid, peracetic acid, citric acid, propionic
acid, etc. The depth of demineralization into the bone surface can
be controlled by adjusting the treatment time, temperature of the
demineralizing solution, concentration of the demineralizing
solution, nature of the demineralizing agent, agitation intensity
during treatment, pressure of the demineralizing environment, and
other forces applied to the demineralizing solution or bone. The
extent of demineralization may be altered or controlled by varying
size of the bone or bone particles being demineralized, by varying
concentration of the demineralization acid, by varying temperature,
by sonicating or applying vacuum during demineralization, or
other.
The demineralized bone is rinsed with sterile water and/or buffered
solution(s) to remove residual amounts of acid and thereby raise
the pH. A suitable defatting/disinfectant solution is an aqueous
solution of ethanol, the ethanol being a good solvent for lipids
and the water being a good hydrophilic carrier to enable the
solution to penetrate more deeply into the bone particles. The
aqueous ethanol solution also disinfects the bone by killing
vegetative microorganisms and viruses. Ordinarily, at least about
10 to 40 percent by weight of water (i.e., about 60 to 90 weight
percent of defatting agent such as alcohol) is present in the
defatting disinfecting solution to produce optimal lipid removal
and disinfection within the shortest period of time. A suitable
concentration range of the defatting solution is from about 60 to
about 85 weight percent alcohol. In one embodiment, the defatting
solution has a concentration of about 70 weight percent
alcohol.
In some embodiments, the demineralized bone comprises surface
demineralized bone. Surface demineralization of bone to a depth
just sufficient to expose the osteons provides bone having improved
biological response while maintaining a mineralized core portion
capable of sustaining mechanical loads. Depth of demineralization
may be defined by size of the particle, amount of time the particle
is in acid solution, concentration of the acid solution, volume of
the acid solution, and/or temperature of the acid solution, and
physical forces applied to the bone.
In some embodiments, the bone may be surface demineralized. The
surface may be an inner surface, such as inside trabeculae or
inside a Haversian canal. In other embodiments the surface may be
an outer surface. In some embodiments, surface demineralized refers
to the bone comprising at least one outer surface, or zone of an
outer surface, that is demineralized and possessing a
non-demineralized core. In some embodiments, the entirety of the
surface may be partially demineralized. In other embodiments, a
portion of the surface may be demineralized, such as by exposing
only a portion of a particle to the demineralization process, by
exposing a portion of the surface to a greater or lesser extent of
the demineralization process, by masking, etc. Demineralization may
be done to a certain percentage. In some embodiments, that
percentage relates to weight percentage. In other embodiments, that
percentage relates to percentage of the size of the bone being
demineralized, or to the depth of demineralization. The depth of
demineralization of the at least one outer surface thus may be
viewed as a percentage of the size of the bone being demineralized
or may be viewed as an absolute number.
Demineralization thus may be carried out to a percentage depth of
the size of the bone being demineralized. FIGS. 4a and 4b
illustrate surface demineralized bone particles. The bone particle
100 of FIG. 4a is substantially spherical. The bone particle 110 of
FIG. 4b is somewhat elongate.
As shown, the bone particle 100 of FIG. 4a has a demineralized
surface region 106 and a non-demineralized core 108. The bone
particle 100 includes a length 102 along its longest dimension and
a length 104 along its shortest dimension. The length 102 in the
longest dimension comprises first and second demineralized portions
103a and 103b and a nondemineralized portion 105. A percentage of
demineralization in the longest dimension may be determined by
summing the length of the first and second demineralized portions
103a and 103b and dividing that total by the length 102 (comprising
103a, 103b and 105). The length 104 in the shortest dimension
likewise comprises first and second demineralized portions 107a and
107b and a nondemineralized portion 109. A percentage of
demineralization in the shortest dimension may be determined by
summing the length of the first and second demineralized portions
107a and 107b and dividing that total by the length 104 (comprising
107a, 107b and 109). A total percentage demineralization may be
determined by averaging the percent demineralization in the longest
dimension with the percent demineralization in the shortest
dimension.
As shown, the bone particle 110 of FIG. 4b has a demineralized
surface region 116 and a non-demineralized core 118. The bone
particle 110 includes a length 112 along its longest dimension and
a length 114 along its shortest dimension. The longest dimension
and shortest dimension are taken as those measuring largest and
smallest, respectively, such as by a micrometer or using other by
suitable manner and generally going through the center of the bone
particle 110. The length 112 in the longest dimension comprises
first and second demineralized portions 113a and 113b and a
nondemineralized portion 115. A percentage of demineralization in
the longest dimension may be determined by summing the length of
the first and second demineralized portions 113a and 113b and
dividing that total by the length 112 (comprising 113a, 113b, and
115). The length 114 in the shortest dimension likewise comprises
first and second demineralized portions 117a and 117b and a
nondemineralized portion 119. A percentage of demineralization in
the shortest dimension may be determined by summing the length of
the first and second demineralized portions 117a and 117b and
dividing that total by the length 114 (comprising 117a, 117b, and
119). A total percentage demineralization may be determined by
averaging the percent demineralization in the longest dimension
with the percent demineralization in the shortest dimension.
Alternatively, percentage demineralization may be based on weight
percent demineralized of total weight of the bone particle.
In some embodiments, demineralization may be carried out to a depth
of, for example, at least about 100 microns. Surface
demineralization may alternatively be done to a depth less than or
more than about 100 microns. Generally, surface demineralization
may be done to a depth of at least 50 microns, at least 100
microns, at least 200 microns, or other. Accordingly, in some
embodiments, the demineralized bone comprises at least one outer
surface possessing at least one demineralized zone and a
non-demineralized core, wherein the demineralized zone of the outer
surface of the bone may be, for example, at least about 100 microns
thick. The demineralized zone may alternatively be less than or
more than about 100 microns thick. The demineralized zone of the
surface of the bone is osteoinductive, and therefore promotes rapid
new ingrowth of native host bone tissue into an osteoimplant
comprising surface demineralized bone. The osteoimplant may
comprise surface demineralized monolithic bone or an aggregate of
surface demineralized bone particles, and may be substantially
solid, flowable, or moldable. The demineralized zone of the surface
of the bone can be any surface portion.
When it is desirable to provide an osteoimplant having improved
biological properties while still substantially maintaining the
strength present in the osteoimplant prior to demineralization, for
example where monolithic bone is used, the extent and regions of
demineralization of the monolithic bone may be controlled. For
example, depth of demineralization may range from at least about
100 microns to up to about 7000 microns or more, depending on the
intended application and graft site. In some embodiments, the depth
of demineralization is between 100 to about 5000 microns, between
about 150 to about 2000 microns, or between about 200 microns to
about 1000 microns. In alternative embodiments, depth of
demineralization may be less than about 100 microns. Reference is
made to U.S. Pat. No. 7,179,299, herein incorporated by reference
for discussion of surface demineralization.
A benefit of surface demineralized bone is that the demineralized
zone(s) can elastically yield under applied force while the
mineralized core has strength and load bearing capacity exceeding
that of demineralized bone. Thus, when the surface demineralized
bone is subjected to an applied load, the demineralized zones can
conform to contours of adjacent bone tissue and thereby minimize
voids or spaces between the osteoimplant and adjacent bone tissue.
This can be useful because host bone tissue will not grow to bridge
large voids or spaces. Thus, by conforming to the contours of
adjacent bone tissue, an osteoimplant comprising surface
demineralized monolithic bone exhibits enhanced biological
properties such as, for example, incorporation and remodeling. The
non-demineralized inner core imparts mechanical strength and allows
the monolithic osteoimplant to bear loads in vivo. Other
non-demineralized zones provide improved tolerances when engaged
with other objects such as, for example, insertion instruments,
other implants or implant devices, etc. It is noted that some of
these characteristics may also be exhibited by an osteoimplant
comprising an aggregate of surface-demineralized bone
particles.
In one embodiment, an osteoinductive composition comprising
partially demineralized (or surface demineralized) bone particles
is provided. The partially demineralized bone particles may, for
example, range in size from 500 .mu.m to 4 mm. In one embodiment
10-80 percent of the mineral of the mineral content of the bone is
removed. When comprised of partially demineralized bone particles,
the osteoinductive composition has a relatively large demineralized
surface area relative to volume. The particulation further
increases the rate of remodeling of the osteoinductive
composition.
Mixtures of one or more types of demineralized bone-derived
elements can be employed. Moreover, one or more of types of
demineralized bone-derived elements can be employed in combination
with non-demineralized bone-derived elements, i.e., bone-derived
elements that have not been subjected to a demineralization
process. Thus, e.g., the weight ratio of non-demineralized to
demineralized (including fully demineralized, partially
demineralized, and surface demineralized) bone elements can broadly
range from less than 0:1 to about 0:1 to about approaching 1:0 or
greater. Further, in some embodiments, mixtures of different types
of bone-derived elements and different levels of
demineralization--for example surface demineralized bone chips or
particles and fully demineralized pressed bone fibers, described
below--may be used. Suitable amounts can be readily determined by
those skilled in the art on a case-by-case basis by routine
experimentation.
As discussed, the bone may be ground or otherwise processed into
particles of an appropriate size before or after demineralization.
For preparing surface demineralized bone particles, the bone is
particulated and then surface demineralized. In certain
embodiments, the particle size is greater than 75 microns, for
example ranging from about 100 to about 3000 microns, or from about
200 to about 2000 or up to greater than 10,000 microns. In some
embodiments, the particle size may be below about 2.8 mm diameter,
or may be between about 2.8 and about 4.0 mm diameter. After
grinding the bone, the mixture may be sieved to select those
particles of a desired size. In certain embodiments, the bone
particles may be sieved though a 50 micron sieve, a 75 micron
sieve, and or a 100 micron sieve.
Alternatively, or additionally, the bone may be particulated to
form elongate particles or fibers. The bone may be particulated in
any suitable manner, such as by milling or pressing. The bone
fibers may comprise threads or filaments having a median length to
median thickness ratio of at least about 10:1 and up to about
500:1, a median length of from about 2 mm to about 400 mm, a medium
width of about 2 mm to about 5 mm, and a median thickness of from
about 0.02 mm to about 2 mm. An osteoinductive composition
comprising bone fibers tends to more readily retain its shape due,
it would appear, to the tendency of the bone particles to become
entangled with each other. The ability of the osteoinductive
composition to maintain its cohesiveness and to resist erosion
subsequent to being applied to an osseus defect site is
advantageous since it enhances utilization of the available bone
particles. Bone fibers whose median length to median thickness
ratio is at least about 10:1 can be readily obtained by any one of
several methods, e.g., shaving the surface of an entire bone or
relatively large section of bone. Another procedure for obtaining
the bone fibers, useful for pieces of bone of up to about 100 mm in
length, is the Cortical Bone Shredding Mill available from Os
Processing Inc., 3303 Carnegie Avenue, Cleveland, Ohio 44115.
Reference is made to U.S. Pat. Nos. 5,314,476, 5,510,396,
5,507,813, and 7,323,193 herein incorporated by reference for
discussion of bone fibers.
After demineralization, water optionally may be removed from the
bone particles [block 37 of FIG. 2] and sterilized [block 39 of
FIG. 2]. Drying may comprise lyophilization, critical point drying,
vacuum drying, solvent dying, or other drying technique. Removing
water from the particles may be referred to as drying the particles
or dehydrating the particles and may be done to any suitable level.
For example, in some embodiments 70% of the water in the bone is
removed, 80% of the water in the bone is removed, 90% of the water
in the bone is removed, 90% of the water in the bone is removed,
95% of the water in the bone is removed, or 98% or more of the
water in the bone is removed.
Sterilization may be done in any suitable manner. In one
embodiment, sterilization may comprise heat sterilizing the bone
without substantially degrading biological properties of the
tissue. In some embodiments, sterilization comprises gentle heating
of the bone. In another embodiment, sterilization comprises heating
the bone in the absence of oxygen. In a further embodiment,
sterilization comprises heating the tissue in the presence of
supercritical CO.sub.2. U.S. patent application Ser. No. 12/140,062
to Method of Treating Tissue, filed Jun. 16, 2008, discloses
methods of sterilization suitable for use with the present
invention and is herein incorporated by reference for the purposes
of all that is disclosed therein.
In some embodiments, the demineralized bone may further be treated,
for example to at least partially remove antigens.
IV. Treat the Bone
In accordance with some embodiments, the demineralized bone may be
treated such that the collagen structure of the bone is disrupted,
shown at block 16 of FIG. 1. Disruption may be done in any suitable
manner including, for example, heat treatment, chemical treatment,
mechanical treatment, energy treatment (e.g., x-ray or radiation),
and others. The collagen structure of bone comprises a triple helix
form. The bone may be treated such that the triple helix form
unwinds but covalent crosslinks of the structure remain intact. In
general, the treatment is such that the collagen in the bone is
denatured or digested to the point where protease enzymes can
readily attack it, while at the same time avoiding the creation of
toxic byproducts, and maintaining some of the original strength of
the bone. Cortical bone treated as provided herein generally
remodel faster than untreated cortical bone, and retain strength in
excess of that of cancellous bone.
More specifically, collagen consists of fibrils composed of
laterally aggregated, polarized tropocollagen molecules (MW
300,000). Each tropocollagen unit consists of three helically wound
polypeptide .alpha.-chains around a single axis. The strands have
repetitive glycine residues at every third position and numerous
proline and hydroxyproline residues, with the particular amino acid
sequence being characteristic of the tissue of origin.
Tropocollagen units combine uniformly to create an axially
repeating periodicity. Cross linkages continue to develop and
collagen becomes progressively more insoluble and resistant to
lysis on aging. Gelatin results when soluble tropocollagen is
denatured, for example on mild heating, and the polypeptide chains
become randomly dispersed. In this state the strands may readily be
cleaved by a wide variety of proteases.
Various methods for disrupting the collagen structure of the
demineralized bone may be used. For example, heat treatment,
treatment with collagenase, other chemical treatment, mechanical
treatment, or energy treatment may be employed. For the purposes of
illustration, discussion is made of treating the bone after it has
been particulated and demineralized. It is to be understood that
the order of particulation, demineralization, and treatment may be
varied. U.S. patent application Ser. No. 12/140,025, for
Osteoinductive Demineralized Cancellous Bone, filed Jun. 16, 2008,
is herein incorporated by reference in its entirety for the
purposes of all that is disclosed therein.
Heat Treatment
In embodiments wherein treating the bone comprises heat treatment
of the bone, the heat treatment may comprise, for example, gentle
heating of the bone. In other embodiments, the heat treatment may
comprise high temperature heating of the bone, heating the bone in
the absence of oxygen, or heating the bone in the presence of
supercritical fluids such as CO.sub.2. Generally, any suitable form
of heat treatment may be used.
Treatment of the partially demineralized bone may comprise heating
the bone to temperatures ranging from approximately 40.degree. C.
to approximately 120.degree. C. for period of time ranging from
approximately 1 minute to approximately 96 hours. Heating may be
done with the partially demineralized bone in a dry state, in
distilled water, in a neutral buffer solution, or other. The
osteoinductive composition may exhibit the ability to induce the
formation of heterotopic bone in a higher order animal such as a
dog, human, or sheep. In some embodiments, the osteoinductive
composition may be combined with osteoinductive growth factors
extracted from bone, recovered from acid used to demineralized
bone, or other.
Thus, in a first embodiment, gentle heating of the bone is
performed to disrupt the collagen structure of the bone. Such
gentle heating denatures proteins in the bone. Heating may be
performed, for example, at temperatures of approximately 60 to
70.degree. C. Gentle heating generally does not chemically degrade
the proteins in the bone. Gentle heating limits potential
inflammatory response. In another embodiment, the bone may be
defatted before the heat treatment to remove lipids, which are a
potential thermal peroxygen compound source. Further, in some
embodiments, water may be removed from the bone before heating (as
at block 39 of FIG. 2).
In another embodiment, the bone is heated in the absence of oxygen.
Heating in the absence of oxygen may be done in any suitable
manner. For example, heating may be done using an inert atmosphere,
a reducing atmosphere, a vacuum, a shielding coating (providing the
coating over the tissue being done during preparation of the
tissue), or other means. Heating cortical bone in the absence of
oxygen produces a faster remodeling cortical bone when implanted in
a vertebrate species, with a strength at least equal to that of
cancellous bone. Generally, cortical bone so treated possesses at
least 30% of its original strength. In some embodiments, the
heating conditions may be selected such that they will result in
virally inactivated bone tissue. For example, the bone may be
heated at temperatures of approximately 100 to 250.degree. C.
In some embodiments of heating in the absence of oxygen, the bone
is heated in an inert atmosphere or in a reducing atmosphere. Such
atmosphere acts as a protective atmosphere. Inert atmospheres may
include argon, nitrogen, helium, CO.sub.2 (including supercritical
CO.sub.2), a hydrocarbon vapor, mixtures of these gases, etc.
Reducing atmospheres may comprise pure hydrogen or hydrogen mixed
with an inert gas wherein the atmosphere comprises between 1 and 99
percent hydrogen. Using a reducing gas, reductive free radicals,
for example from hydrogen, are produced to protect against the
effects of oxidative free radicals. In various embodiments, the
bone may be treated in a chamber wherein the protective atmosphere
is introduced to the chamber and released after treatment. The
method of release of the atmosphere may be controlled to affect the
bone. For example, slow release of the atmosphere has little effect
on the bone. In contrast, fast release of the atmosphere may cause
the bone to expand and develop pores.
A further embodiment of heating in the absence of oxygen comprises
coating the bone with a protective thermal coating. The protective
thermal coating forms an oxygen barrier and, thus, the bone with
the protective thermal coating may be heated in an oxygenated
atmosphere. Such protective thermal coating may comprise, for
example, a polymer or wax that does not react with the tissue and
that forms an oxygen barrier. In one embodiment, the protective
coating comprises PolyDTE polymer. In another embodiment, the
protective coating comprises a mix of Poly(lactide-co-glycolide)
and Poly(ethylene glycol). The protective coating may be layered
over a monolithic piece of bone or may be mixed with smaller bone
elements--such as particulated bone. When mixed with particulated
bone, for example, the polymer/bone mix may be molded to form an
implant.
In some embodiments, the bone is surface demineralized and then
incubated in a phosphate buffer. The demineralized surface of the
bone remains osteoinductive. The surface-demineralized bone may
then be heated without addition of enzyme inhibitors (sodium azide
and iodacetic acid).
Reference is made to U.S. patent application Ser. No. 12/140,025,
entitled "Osteoinductive Demineralized Cancellous Bone", filed Jun.
16, 2008, and to U.S. patent application Ser. No. 12/140,062,
entitled "Method of Treating Tissue", filed Jun. 16, 2008, both
herein incorporated by reference for discussion of disrupting the
collagen structure of bone.
Chemical Treatment
In accordance with other embodiments, treating the bone to degrade
the collagen structure of the bone comprises treating the bone with
a chemical. In some embodiments, a chemical may be used to cleave
simultaneously across all three chains of the collagen helix or to
attack a single strand of the collagen helix. In some embodiments,
the chemical cleaves Type I collagen, e.g., degrades the helical
regions in native collagen, preferentially at the Y-Gly bond in the
sequence Pro-Y-Gly-Pro-, where Y is most frequently a neutral amino
acid. This cleavage yields products susceptible to further
peptidase digestion. Any chemical or protease having one or more of
these activities may be used to treat the demineralized bone.
In one embodiment, the bone is treated with a collagenase enzyme.
Generally, when bone is treated with collagenase, natural
degradation products are formed. Because the dense structure of the
bone that inhibits remodeling may complicate an enzyme treatment
process, getting the enzyme to penetrate the bone can be difficult.
Physical methods such as centrifugation in an enzyme solution, or
the use of a solvent such as DMSO, may thus be used.
Collagenases and their activity on collagens of various types have
been extensively studied. A number of collagenase preparations are
available from Worthington Biochemical Corporation, Lakewood, N.J.
In general, a variety of different collagenases known in the art
can be used to disrupt the collagen structure of the bone.
Collagenases are classified in section 3.4.24 under the
International Union of Biochemistry and Molecular Biology
(NC-IUBMB) enzyme nomenclature recommendations (see, e.g.,
3.4.24.3, 3.4.24.7, 3,4.24.19). The collagenase can be of
eukaryotic (e.g., mammalian) or prokaryotic (bacterial) origin.
Bacterial enzymes differ from mammalian collagenases in that they
attack many sites along the helix.
It will be appreciated that crude collagenase preparations contain
not only several collagenases, but also a sulfhydryl protease,
clostripain, a trypsin-like enzyme, and an aminopeptidase. This
combination of collagenolytic and proteolytic activities is
effective at breaking down intercellular matrices, an essential
part of tissue disassociation. Crude collagenase is inhibited by
metal chelating agents such as cysteine, EDTA, or o-phenanthroline,
but not DFP. It is also inhibited by .alpha.2-macroglobulin, a
large plasma glycoprotein. Ca.sup.2+ is required for enzyme
activity. Therefore, it may be desirable to avoid collagenase
inhibiting agents when treating bone matrix with collagenase. In
addition, although the additional proteases present in some
collagenase preparations may aid in breaking down tissue, they may
also cause degradation of desired matrix constituents such as
growth factors. Therefore, a purified collagenase that contains
minimal secondary proteolytic activities along with high
collagenase activity may be used. For example, a suitable
collagenase preparation may contain at least 90%, at least 95%, at
least 98%, or at least 99% collagenase by weight. The preparation
may be essentially free of bacterial components, particularly
bacterial components that could cause inflammatory or immunological
reactions in a host, such as endotoxin, lipopolysaccharide, etc.
Preparations having a purity greater than 99.5% can also be used. A
suitable preparation is chromatographically purified CLSPA
collagenase from Worthington Biochemical Corporation. Various
protease inhibitors may be included that do not inhibit collagenase
but that inhibit various proteases that digest BMP. For example,
protease inhibitors that are known to protect BMP activity from
degradation include N-ethyl maleimide, benzamidine hydrochloride,
iodoacetic acid, PMSF, AEBSF, E-64. Bestatin may also be used,
particularly if the preparation contains aminopeptidase activity.
Any of these protease inhibitors (or others) may be provided in a
composition that is used to treat the demineralized bone.
Bone morphogenetic protein I (BMP-1) is a collagenolytic protein
that has also been shown to cleave chordin (an inhibitor of BMP-2
and BMP-4). Thus, BMP-I may be of use to alter the physical
structure of the demineralized bone (e.g., by breaking down
collagen) and/or to cleave specific inhibitory protein(s), e.g.,
chordin or noggin. Proteins related to any of the proteases
described herein, i.e., proteins or protein fragments having the
same cleavage specificity, can also be used. It will be appreciated
that variants having substantial sequence identity to naturally
occurring protease can be used. For example, variants at least 80%
identical over at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, or 100% of the length of naturally occurring
protease (or any known active fragment thereof that retains
cleavage specificity) when aligned for maximum identity allowing
gaps can be used.
Collagen can also be broken down by treatment with a strong base,
such as sodium hydroxide. Thus, in some embodiments, sodium
hydroxide can be introduced to the bone to disrupt the collagen
structure of the bone. Such introduction may be in the form of a
solution with penetration aided by a centrifuge and/or the addition
of DMSO, as is the case for an enzyme. The base will not harm the
mineral component of bone; so much of the strength (especially
compressive strength) is maintained.
Other chemicals, such as cyanogen bromide, may alternatively be
used to alter the collagen structure of the bone.
Combinations of treatments designed to degrade collagen can be
used; for example, a mild heating combined with an enzyme or base
treatment; or an enzyme treatment followed by a radiation
treatment. Any suitable combination of treatments, including
treatments not discussed herein, may be used.
In some embodiments, the partially demineralized bone, whether
provided as an aggregate of particles or a monolithic bone, may be
compressed to increase its density. The structure of cancellous
bone is less dense than that of cortical bone. By compressing the
structure of the cancellous bone, the osteoinductive potential is
increased. Compression may be done before or after addition of an
extract and/or carrier to the partially demineralized bone.
Compression may be achieved via any suitable mechanism. For
example, compression may be achieved by mechanical means, heat, or
chemical modification of the collagenous structure. Reference is
made to U.S. patent application Ser. No. 11/764,026, entitled
"Osteoinductive Demineralized Cancellous Bone", filed Jun. 15,
2007, herein incorporated by reference for discussion of techniques
for compressing the partially demineralized bone.
V. Add Demineralized Bone Matrix
In some embodiments, demineralized bone matrix (DBM) may be added
to the partially demineralized bone particles. The DBM may comprise
monolithic bone, bone particles, bone fibers, or other composition
of bone. Any suitable manner may be used to add the demineralized
bone matrix to the partially demineralized bone particles. Any
suitable ratio of demineralized bone matrix to partially
demineralized bone particles may result. The various processing
steps set forth herein may be performed in any suitable sequence
that provides the desired results. For example, in some
embodiments, the at least partially demineralized bone particles
are processed, for example dried, and the demineralized bone matrix
is processed, for example dried, separately from the partially
demineralized bone particles. In these embodiments, the at least
partially demineralized particles and the demineralized bone matrix
are combined after processing. In other embodiments, the partially
demineralized bone particles and the demineralized bone matrix may
be combined and then processed, for example, dried, together. Other
steps also may be performed in different orders, combined, or
omitted, within the spirit of the present invention.
In one embodiment, the DBM comprises pressed DBM fibers. Pressed
DBM fibers may comprise elongate bone particles. The elongate bone
particles or bone fibers may comprise threads or filaments having a
median length to median thickness ratio of at least about 10:1 and
up to about 500:1, a median length of from about 2 mm to about 400
mm, a medium width of about 2 mm to about 5 mm, and a median
thickness of from about 0.02 mm to about 2 mm. The DBM fibers may
be pressed bone fibers.
Pressed bone fibers refers to the manner by which the bone fibers
are formed. Generally, forming the bone fibers by pressing the
bone, as described below, results in intact bone fibers of longer
length than other methods of producing elongate bone fibers, with
the bone fibers retaining more of the native collagen structure.
The bone may be particulated via pressure applied to the bone, as
discussed in U.S. Pat. No. 7,323,193.
The entire bone can then be demineralized or can be sectioned
before demineralization. The entire bone or one or more of its
sections is subjected to demineralization to reduce the inorganic
content of the bone, e.g., to less than about 10% by weight, less
than about 5% by weight, or less than about 1% by weight, residual
calcium. Demineralization of the bone can be accomplished in
accordance with known and conventional procedures, as described
above.
Following demineralization, the bone is subdivided into
demineralized bone fibers of desired configuration and size. One
method suitable for subdividing demineralized bone stock is to
subject the bone to pressing. One pressing technique comprises
applying pressure to the unconstrained demineralized bone. Examples
include pressing the bone using a mortar and pestle, applying a
rolling/pressing motion such as is generated by one or more rolling
pins, or pressing the bone pieces between flat or curved plates. In
other embodiments, flat or any other suitable configuration of
plate or pressing surface may be used. These flattening pressures
cause the bone fibers to separate. Pressing demineralized bone in
this manner provides intact natural bone collagen fibers (as
opposed to composite fibers made from joined short fiber sections)
that can be as long as the fibers in the demineralized bone stock
from which they were obtained.
Another suitable pressing technique comprises mechanically pressing
demineralized bone which is constrained within a sealed chamber
having at least one aperture in its floor or bottom plate. The
separated fibers extrude through the holes with the hole diameter
limiting the maximum diameter of the extruded fibers. As with the
unconstrained pressing method, this constrained technique results
in fibers that are largely intact (as far as length is concerned)
but separated bone collagen bundles.
In a combined unconstrained/constrained pressing technique that
results in longer fibers by minimizing fiber breakage, the
demineralized bone is first pressed into an initially separated
mass of fibers while in the unconstrained condition and thereafter
these fibers are constrained within the sealed chamber where
pressing is continued.
In general, pressing of demineralized bone to provide demineralized
bone fibers can be carried out at from less than about 1,000 psi,
to about 1,000 to about 40,000 psi, or from about 5,000 to about
20,000 psi, or greater than about 40,000 psi.
Depending on the procedure employed, the demineralized bone fibers
may comprise elongate bone fibers with at least about 80 weight
percent, at least about 90 weight percent, or at least about 95
weight percent, of the fibers possessing a median length of from
about 2 to about 300 mm or greater, for example, a median length of
from about 5 to about 50 mm, a median thickness of from about 0.5
to about 15 mm, for example, a median thickness of from about 1 to
about 5 mm, a median width of from about 2 to about 35 mm, for
example, a median width of from about 2 to about 20 mm, and a
median length to thickness ratio and/or a median length to width
ratio of from about 2 to 200, for example from about 10 to about
100. In some embodiments, the mass of bone fibers can be graded or
sorted into different sizes, e.g., by screening, and/or any less
desirable size(s) of bone fibers that may be present can be reduced
or eliminated.
The demineralized bone fibers may be dried, for example using
lyophilization, critical point drying, vacuum drying, solvent
dying, or other drying technique.
VI. Provide a Tissue-Derived Extract
Returning to FIG. 1, a tissue-derived extract optionally may be
added, shown at block 18, to the partially demineralized bone, or,
in some embodiments, to the partially demineralized bone and
demineralized bone matrix. The extract may be derived from any
suitable tissue, such as bone, bladder, kidney, brain, skin, or
connective tissue. Further, the extract may be derived in any
suitable manner. The extract may be allogeneic, autogeneic,
xenogeneic, or transgenic. In embodiments wherein the extract is
bone-derived, the bone may be cortical, cancellous, or
corticocancellous and may be demineralized, partially
demineralized, or mineralized. In some embodiments, the extract may
comprise demineralized bone, partially demineralized bone, mineral
derived from bone, or collagen derived from bone. In some
embodiments, the tissue-derived extract may be a protein
extract.
As previously discussed, in the art, demineralized bone is often
particulated. Typically, such particulation comprises sieving the
particles to select only particles having at least a certain size.
Particles below that size fall through the sieve and are
categorized as waste particles. In accordance with some
embodiments, the extract is derived from such waste particles.
DBM preparations have been used for many years in orthopedic
medicine to promote the formation of bone. For example, DBM has
found use in the repair of fractures, in the fusion of vertebrae,
in joint replacement surgery, and in treating bone destruction due
to underlying disease such as rheumatoid arthritis. DBM is thought
to promote bone formation in vivo by osteoconductive and
osteoinductive processes. The osteoinductive effect of implanted
DBM compositions is thought to result from the presence of active
growth factors present on the isolated collagen-based matrix.
A simple and economically viable method for extracting
osteoinductive factors from bone is provided herein. It is to be
appreciated that this method may be applied to other tissues. The
method comprises extracting osteoinductive factors such as
noncollagenous proteins (including osteogenic growth factors) from
DBM using a chaotropic solvent or a detergent. The chaotropic
solvent may be guanidine hydrochloride of any suitable
concentration, such as 4M. The detergent may be sodium
dodecylsulfate in any suitable concentration, such as 1%. The
chemical used for extraction is removed in an efficient manner that
preserves the biological activity of the growth factors. The
biologically active components are concentrated by purifying away
nonessential proteins and inhibitors of bone morphogenetic protein,
and the protein extracts are then combined with a biologically
compatible delivery vehicle.
Using the method described, the extraction process is optimized by
using relatively low cost chaotropic agents, and relatively
easy-to-remove detergents. Methods to increase the speed of
renaturing the extracted proteins are further provided. Typically
in the art, dialysis against water is used to remove the detergent
or chaotropic agent. However, by precipitating the proteins with
ethanol, acetone, ammonium sulfate, or polyethylene glycol,
dialysis against water is not necessary. Further, ultrafiltration
may be used, thereby also avoiding dialysis.
Generally, extracted osteoinductive factors have lower specific
bone forming activity when compared to the starting material (e.g.,
the tissue from which the osteoinductive factors are extracted).
This may be caused by protein denaturation that results from
extraction. For example, when guanidine is used to extract
hydrophobic osteoinductive proteins, the proteins lose their native
three-dimensional conformation. As a result, unless they regain
their normal shape upon removal of the guanidine, they no longer
are active. The addition of chemical chaperones to the guanidine
solution may prevent this protein denaturation. Suitable chemical
chaperones include glycerol, trehalose, proline, glycine betaine,
and dextrose, along with mixtures of these and others. These
chemical chaperones enable the osteoinductive proteins to regain
their native three-dimensional conformation when the guanidine is
removed. They also substantially prevent protein denaturation
during lyophilization.
A method for extracting osteoinductive factors from the mineral
component of bone is provided to recover growth factor activity
that is normally lost during the demineralization process. It is
known that 4 M guanidine hydrochloride can extract osteoinductive
factors from finely powdered mineralized bone. Additionally,
osteoinductive factors can be recovered from the acid that is
typically used to demineralized bone. These osteoinductive factors
are normally lost during the demineralization process and treated
as waste.
In some embodiments, the tissue-derived extract to be added to the
partially demineralized bone may be derived from the acid used to
demineralize bone. Growth factors may be extracted from the mineral
phase of bone using, for example, the following procedure. As
previously described, bone is at least partially demineralized. The
bone may comprise powder, fibers, chips, or other. The bone may be
demineralized in an acid, for example 1M citric acid, 2M citric
acid, or 0.6N HCl, at temperatures ranging from, for example
1.degree. C. to 28.degree. C. for time period of for example 10
minutes to 96 hours. In one embodiment, the bone is demineralized
in an acid at a temperature of 4.degree. C. After demineralization,
the acid used for demineralization contains growth factors and
mineral. The acid may be dialyzed against water to cause the
mineral phase and the protein growth factors to co-precipitate.
This biphasic (protein and mineral) material may then be collected
by filtration or centrifugation and combined with a carrier or
lyophilized.
In alternative embodiments, the protein and mineral material in the
acid may be separated by dialyzing the acid, also referred to as
the demineralization bath, against a weak acid, for example 0.25M
citric acid. In such embodiment, the mineral phase passes through
the dialysis bag and the protein phase (collagen fragments, growth
factors, etc.) is left within the bag. The protein phase can then
be recovered by dialyzing against water and separating water
soluble and water insoluble proteins from one another.
In one embodiment, the method for extracting growth factors
comprises demineralizing powdered bone with dilute acid within a
dialysis bag. Suitable dilute acid includes 0.05 M to 1.0 M HCl and
1M or 2M citric acid. After removing the demineralized bone, the
contents of the bag may be further dialyzed against dilute acid to
remove the mineral components. A volatile acid, such as acetic
acid, can be used to facilitate recovery by lyophilization.
Proteases may reduce the activity of the osteoinductive factors in
demineralized bone by breaking down those osteoinductive factors.
This negative effect may be reduced or eliminated by adding
protease inhibitors to the HCl solution. Suitable protease
inhibitors include N-ethyl maleimide, benzamidine HCl, cysteine, or
iodoacetic acid. Alternatively, the bone may be heated briefly to
inactivate the proteases, which are relatively more heat sensitive
than the growth factors. A suitable heating regimen is 5 minutes at
60.degree. C., or 1 minute at 90.degree. C.
Thus, mineralized bone or bone mineral recovered from
demineralization acid may be used for purifying recovered proteins.
The protein phase recovered from the demineralization bath may be
solubilized in urea or other form of detergent solution. The bone
stimulating growth factors may then be purified, for example using
a hydroxyapatite affinity chromatography scheme.
In one embodiment the tissue derived extract may comprise a protein
composition substantially free from inorganic components. The
protein composition may comprise less than 5% inorganic components
by weight. In an alternative embodiment, a protein composition
comprising organic components ranging from approximately 6% to
approximately 20% by weight is provided. In another embodiment, a
protein composition comprising organic components ranging from
approximately 21% to approximately 50% may be provided. In yet a
further embodiment, a protein composition comprising organic
components ranging from approximately 51% to approximately 90% may
be provided. The protein composition may be recovered from acid
used to demineralize bone. The protein composition may
alternatively be extracted from other tissues or in other manners.
The proteinaceous material of the protein composition may be
purified by chromatography, electrophoresis, or other chemical or
physical means. The protein composition may be combined with
another material such as demineralized bone, hydroxyapatite,
tricalcium phosphate (TCP), dicalcium phosphate (DCP), or other. In
some embodiments, the protein composition may exhibit the ability
to induce heterotopic bone formation in an athymic animal. In some
embodiments the protein composition can serve as a source of
collagen Type I, collagen Type I residues, and other extracellular
matrix proteins that can support tissue repair processes such as
angiogenesis, osteoconduction and wound healing. As the protein
material has desirable handling properties when combined with water
or glycerol, the protein can also serve as a carrier for a variety
of bone forming matrices including partially demineralized or fully
demineralized bone matrix.
In some embodiments, the tissue-derived extract may be solubilized
in an appropriate medium, such as 6M urea, exposed to
hydroxyapatite, TCP, DCP, mineralized bone, surface demineralized
bone, or mineral recovered from acid used to demineralize bone. The
protein may further be permitted to adsorb onto mineral surfaces
and be washed with a solution comprising, for example, sodium
phosphate ranging from approximately 1 mM to 50 mM in
concentration. The proteins may then be eluted with a solution
comprising, for example, sodium phosphate ranging in concentrations
from between approximately 100 mM to approximately 500 mM.
With specific reference to extracts from bone, proteins in bone
matrix tend to be insoluble and may associate with the bone matrix.
Generally, collagens are among the most insoluble osteoinductive
factors. Extraction methods may be used to increase the solubility
of the osteoinductive factors to facilitate extraction of the
osteoinductive factors. Generally, growth factors are hydrophobic
and are not readily soluble. Thus, growth factors may be treated to
improve solubility.
The solubility of demineralized bone in one or more solvents (e.g.,
an aqueous medium) may be changed, e.g., increased, relative, for
example, to the solubility of a standard demineralized bone not
exposed to the treatment. Preferably, the aqueous medium is at
physiological conditions, e.g., pH, osmotic pressure, salt
concentration, etc. within physiologically appropriate ranges. For
example, the pH may be approximately 7.2-8.0, or preferably
7.4-7.6. The osmotic pressure may be approximately 250-350 mosm/kg,
280-300 mosm/kg, etc. More generally, the pH may be between
approximately 3-11, 4-10, 5-9, 6-8.5, etc. The osmotic pressure may
be between 50-500 mosm/kg, 100-350 mosm/kg, etc. The salt
concentration may be approximately 100-300 mM NaCl, e.g.,
approximately 150 mM NaCl. The aqueous medium may be tissue culture
medium, blood, extracellular fluid, etc., and the physiological
conditions may be conditions such as are typically found within
these fluids and/or within a body tissue such as muscle. The
solubility may be increased at any temperature, e.g., room
temperature, body temperature of a subject such as a human or
animal, etc.
Collagenase treatment of standard human DBM increases its
solubility relative to that of untreated standard human DBM. The
solubility of the DBM may be increased by exposure to an
appropriate treatment or condition, e.g., collagenase treatment,
radiation, heat, etc. The extent to which the solubility is
increased may be varied by varying the nature of the treatment
(e.g., the enzyme concentration) and/or the time over which it is
applied. A combination of treatments may be used. In certain
embodiments, the solubility of the DBM composition is greater than
that of a standard DBM composition by between 10% and 4000%
percent. For example, the solubility may be greater by between 10%
and 100%, 100% and 500%, 500% and 1000%, 1000% and 2000%, 2000% and
3000%, 3000% and 4000% or any other range between 10% and 4000%.
The solubility may be assessed at any time following the treatment
to increase the solubility of the DBM composition. For example, the
DBM may be placed in aqueous medium for a period of time such as
24-48 hours, 3, 4, 5, 6, or 7 days, 10 days, 14 days, etc. The
amount of DBM remaining after the period of time is quantitated
(e.g., dry weight is measured) and compared with the amount that
was present initially. The extent to which the amount decreases
after a period of time serves as an indicator of the extent of
solubilization.
In alternative embodiments, tissue-derived extracts may be derived
in any suitable manner. Further, during extraction, coprecipitates
may be used. Thus, for example, using bone, the bone may be treated
with a chaotropic solvent such as guanidine hydrochloride. The bone
and chaotropic solvent are dialyzed against water. As the
chaotropic solvent decreases, it is replaced by water. Precipitates
are then extracted. Coprecipitates, such as protein, collagen,
collagen fragments, albumen, or protein with RGD sequences, may be
extracted. The extracted osteoinductive factors and coprecipitates
may then be blended into a homogenous mixture.
In one embodiment, a simplified extraction process may be used that
is amenable to batch processing. K. Behnam, E. Brochmann, and S.
Murray; Alkali-urea extraction of demineralized bone matrix removes
noggin, an inhibitor of bone morphogenetic proteins; Connect Tissue
Res. 2004, 45(4-5):257-60.
A number of naturally occurring proteins from bone or recombinant
osteoinductive factors have been described in the literature and
are suitable for use in the osteoinductive composition as a
tissue-derived extract. Recombinantly produced osteoinductive
factors have been produced by several entities. Creative
Biomolecules of Hopkinton, Mass., produces an osteoinductive factor
referred to as Osteogenic Protein 1, or OP1. Genetics Institute of
Cambridge, Mass., produces a series of osteoinductive factors
referred to as Bone Morphogenetic Proteins 1-13 (i.e., BMP 1-13),
some of which are described in U.S. Pat. Nos. 5,106,748 and
5,658,882 and in PCT Publication No. WO 96/39,170, each herein
incorporated by reference. Purified osteoinductive factors have
been developed by several entities. Collagen Corporation of Palo
Alto, Calif., developed a purified protein mixture that is
purported to have osteogenic activity, as described in U.S. Pat.
Nos. 4,774,228, 4,774,322, 4,810,691, and 4,843,063, each herein
incorporated by reference. Urist developed a purified protein
mixture which is purported to be osteogenic, as described in U.S.
Pat. Nos. 4,455,256, 4,619,989, 4,761,471, 4,789,732, and
4,795,804, each herein incorporated by reference. International
Genetic Engineering, Inc. of Santa Monica, Calif., developed a
purified protein mixture that is purported to be osteogenic, as
described in U.S. Pat. No. 4,804,744, herein incorporated by
reference.
One osteoinductive factor that may be used as a tissue-derived
extract in the osteoinductive composition is described in detail in
U.S. Pat. No. 5,290,763, herein incorporated by reference. This
osteoinductive factor has a high osteogenic activity and degree of
purity. The osteoinductive factor of the '763 patent exhibits
osteoinductive activity at about 3 micrograms when deposited onto a
suitable carrier and implanted subcutaneously into a rat. In one
embodiment, the osteoinductive factor is an osteoinductively active
mixture of proteins that exhibit the gel separation profile shown
in FIG. 1 of U.S. Pat. No. 5,563,124, herein incorporated by
reference.
In some embodiments, the tissue-derived extract may comprise bone
stimulating growth factors, for example recovered from the mineral
phase of bone. The bone stimulating growth factors may be purified
using an apatite affinity chromatography scheme. Thus, mineralized
or surface demineralized bone may be used as a chromatography
resin. Bone mineral comprises calcium phosphate sales similar to
hydroxyapatite. To use mineralized or surface demineralized bone as
a chromatography resin, excess lipid and protein may be removed
from the surfaces of the bone. In other embodiments, a similar
scheme may be done using demineralized bone matrix as a resin. In
yet further embodiments, recovered inorganic bone mineral (sintered
or unsintered) may be used as the chromatography resin.
In one embodiment, the protocol for such scheme may be as follows.
Mineralized bone particles, for example ranging from 100 .mu.m to 5
mm, are prepared. The surface of the mineralized bone particles is
cleaned, for example by soaking or stirring the bone particles in a
dilute base such as 0.1M NaOH for several minutes. Generally, such
surface cleaning removes proteins as well as lipids. In alternative
embodiments, surface cleaning may be performed using supercritical
CO.sub.2. Growth factor extracts from the mineral phase may be
solubilized in a chaotropic solvent such as 6M urea. The growth
factor solution may then be mixed with the mineralized bone
particles, for example, for several minutes. During such mixing,
proteins having an affinity for hydroxyapatite bind to the bone
surfaces. The bone-protein complex is then precipitated and the
supernatant removed. The bone-protein complex may be treated to
remove weakly bound proteins such as collagen fragments while
retaining osteoinductive proteins (the osteoinductive proteins
remain bound to the material). Such treatment may comprise treating
the bone-protein complex with a 6M urea containing low
concentrations of sodium phosphate. The treated bone-protein
complex may be centrifuged and the supernatant aspirated. In some
embodiments, the bone-protein complex may be treated with urea
containing higher concentrations of sodium phosphate (e.g., 100 mM,
180 mM, or 250 mM) to release bound osteoinductive proteins.
Alternatively, the bone-osteoinductive protein complex may be
lyophilized and formulated with a carrier, for example for
orthopedic applications. Further, the bone protein complex may be
used as a growth factor microcarrier that can be distributed in a
DBM macrocarrier.
Extraction may extract, for example, both osteoinductive factors
and their inhibitors. If the inhibitors are extracted, the
osteoinductive factors may be separated out. This may be referred
to as removal of the inhibitors or concentration of the
osteoinductive factors. As a general matter, both the
osteoinductive factors and the inhibitors may be extracted and both
the osteoinductive factors and the inhibitors may be used for
forming the osteoinductive composition. Alternately, only the
osteoinductive factors (and not their inhibitors) are extracted and
only the osteoinductive factors are used for manufacturing the
osteogenic osteoimplant. Lastly, both the osteoinductive factors
and the inhibitors may be extracted and only the osteoinductive
factors may be used for forming the osteoinductive composition. In
some embodiments, it may be desirable to remove inhibitors or
concentrate the osteoinductive factors. This is optional and may be
done by any suitable method. Generally, it may be desirable to
remove the inhibitors quickly without denaturing the osteoinductive
factors. Reference is made to U.S. patent application Ser. Nos.
11/555,606 and 11/555,608, to which the present application claims
priority and which is herein incorporated by reference for
discussion of other processing that may be used. The embodiment of
extraction and resultant use of osteoinductive factors with or
without inhibitors is not a limiting feature of the present
invention.
In some embodiments, the tissue-derived extract may be modified in
one or more ways, e.g., its protein content can be augmented or
modified as described in U.S. Pat. Nos. 4,743,259 and 4,902,296,
the contents of which are incorporated by reference herein. The
extract can be admixed with one or more optional substances such as
binders, fillers, fibers, meshes, substances providing radiopacity,
plasticizers, biostatic/biocidal agents, surface active agents, and
the like, prior to, during, or after adding to the carrier.
VII. Add Extract to the Partially Demineralized Bone
As shown at block 18 of FIG. 1, the tissue-derived extract may be
added to the partially demineralized bone, or, in some embodiments,
to the partially demineralized bone and demineralized bone matrix.
Such addition may be done in any suitable manner. As discussed, the
tissue-derived extract may comprise extracted osteoinductive
factors and possibly inhibitors. For ease of reference, unless
otherwise noted, reference to osteoinductive factors refers to
osteoinductive factors with or without inhibitors.
The tissue-derived extract may be added in any suitable extract
dose. Generally the dosage may be from less than 1.times. to
approximately 10.times.. For the purposes of this disclosure,
1.times. is defined as the amount of extract that may be derived
from a single clinically relevant unit of tissue. For example,
using bone as the tissue, for a 10 cc unit of DBM, mineralized
bone, or surface demineralized bone, 1.times. is the amount of
extract that can be derived from 10 cc of the bone.
When the extract is added to the partially demineralized bone, the
partially demineralized bone may first act as a bulking means for
applying a small amount of extracted material. The partially
demineralized bone also may serve as a scaffold, and may aid in
controlling release kinetics. Any suitable shape, size, and
porosity of partially demineralized bone may be used. Rat studies
show that the new bone is formed essentially having the dimensions
of the device implanted. Generally, particle size influences the
quantitative response of new bone; particles between 70 .mu.m and
420 .mu.m elicit the maximum response. However, other particle
sizes may be used.
The partially demineralized bone may comprise a DBM preparation.
Generally, the DBM preparation will include at least some portion
of surface demineralized bone. DBM prepared by any method may be
employed, including particulate or fiber-based preparations,
mixtures of fiber and particulate preparations, fully or partially
demineralized preparations, mixtures of fully and partially
demineralized preparations, and surface demineralized preparations.
See U.S. Pat. No. 6,326,018, Reddi et al., Proc. Natl. Acad. Sci.
USA (1972) 69:1601-1605; Lewandrowski et al., Clin. Ortho. Rel.
Res., (1995) 317:254-262; Lewandroski et al., J. Biomed. Mater.
Res. (1996) 31:365-372; Lewandrowski et al. Calcified Tiss. Int.,
(1997) 61:294-297; Lewandrowski et al., I Ortho. Res. (1997)
15:748-756, each of which is incorporated herein by reference.
Suitable demineralized bone matrix compositions are described in
U.S. Pat. No. 5,507,813, herein incorporated by reference. As
discussed, the bone may be particulated. In alternative
embodiments, the bone may be in the form of a section that
substantially retains the shape of the original bone (or a portion
thereof) from which it was derived. Also useful are preparations
comprising additives or carriers such as polyhydroxy compounds,
polysaccharides, glycosaminoglycan proteins, nucleic acids,
polymers, poloxamers, resins, clays, calcium salts, and/or
derivatives thereof.
As discussed, the tissue-derived extract may be combined with the
partially demineralized bone. The manner by which the
tissue-derived extract is combined with the partially demineralized
bone can influence the biological activity of the final
composition. The tissue-derived extract may be lyophilized,
resulting in a powder. In some situations, adding a powder to a
bone matrix may be challenging. Thus, it may be desirable to
process a powdered tissue-derived extract to form a homogenous
mixture that may be more easily added to partially demineralized
bone. This can impact release kinetics of any growth factors.
Thus, in a specific example, if the tissue-derived extract is
lyophilized and then added to the partially demineralized bone, the
solution may be inhomogeneous, with most of the tissue-derived
extract concentrated on the outside of the partially demineralized
bone. If the tissue-derived extract is added to very thin DBM
sheets and each sheet is folded in on itself, the distribution of
tissue-derived extract may be more homogenous. The sheets in such
an embodiment can be very thin, on the order of microns. The sheets
may comprise, for example, the partially demineralized bone mixed
with a carrier, described more fully below.
Any suitable method for adding, or dispersing, the tissue-derived
extract to the partially demineralized bone may be used. Generally,
the procedures used to formulate or disperse the tissue-derived
extract onto the partially demineralized bone are sensitive to the
physical and chemical state of both the tissue-derived extract and
the partially demineralized bone. In some embodiments, the extract
may be precipitated directly onto the partially demineralized
bone.
In one embodiment, the tissue-derived extract is blended with a
bulking agent to form a homogenous mixture. This mixture is added
to the partially demineralized bone. Alternatively, the
tissue-derived extract may be blended with coprecipitates and this
blend may be added to the partially demineralized bone.
In some embodiments, after the extract has been added to the
partially demineralized bone, the partially demineralized bone may
have a BMP content (BMP-2 content, BMP-4 content, BMP-7 content,
TGF-beta content, IGF-II content, MMP-13 content, and/or aggregate
BMP content) of at least approximately 110% that of demineralized
bone without added tissue-derived extract.
Thus, in some embodiments, an osteoinductive composition comprising
surface demineralized bone particles and tissue-derived extract is
provided. The tissue-derived extract may be adsorbed to the
surfaces of the partially demineralized bone particles. Weakly
bound components may be eluted using, for example, low
concentrations of sodium phosphate (for example, 5 mM to 50 mM),
thereby concentrating the tissue-derived extract. For extract
derived from bone, in some embodiments, analysis of the proteins
bound to the surfaces of the surface demineralized bone particles
indicates a ratio of Histone H2A to total protein bound elevated by
a factor of 2 to 10,000 times over the normal ratio found in
extracts of demineralized bone matrix or protein recovered from
acid used to demineralize bone. In some embodiments, analysis of
the proteins bound to the surfaces of the surface demineralized
bone particles indicates a ratio of Secreted Phosphoprotein 24 to
total protein bound elevated by a factor of 2 to 10,000 times over
the normal ratio found in extracts of demineralized bone matrix or
protein recovered from acid used to demineralize bone. In some
embodiments, analysis of the proteins bound to the surfaces of the
surface demineralized bone particles indicates a ratio of BMP-2 to
total protein bound elevated by a factor of 2 to 10,000 times over
the normal ratio found in extracts of demineralized bone matrix or
protein recovered from acid used to demineralize bone. In some
embodiments, analysis of the proteins bound to the surfaces of the
surface demineralized bone particles indicates a ratio of BMP-4 to
total protein bound elevated by a factor of 2 to 10,000 times over
the normal ratio found in extracts of demineralized bone matrix or
protein recovered from acid used to demineralize bone. In some
embodiments, analysis of the proteins bound to the surfaces of the
surface demineralized bone particles indicates a ratio of TGF-Beta
to total protein bound elevated by a factor of 2 to 10,000 times
over the normal ratio found in extracts of demineralized bone
matrix or protein recovered from acid used to demineralize
bone.
In some embodiments, no tissue-derived extract may be added to the
partially demineralized bone.
VIII. Add Partially Demineralized Bone to Delivery Vehicle
As shown at block 19 of FIG. 1, the partially demineralized bone,
with or without a tissue-derived extract and/or demineralized bone
matrix, optionally may be used with a delivery vehicle. In one
embodiment, such delivery vehicle may be a carrier to which the
partially demineralized bone is added [block 20 of FIG. 1]. In
another embodiment, such delivery vehicle may be a covering in
which the partially demineralized bone is provided [block 22 of
FIG. 1]. In other embodiments, a carrier and a covering both may be
used. The partially demineralized bone and delivery vehicle
together form an osteoimplant useful in clinical applications.
Add Partially Demineralized Bone to Carrier
The carrier may be formulated to impart specific handling
characteristics to the composition. For example, in some
embodiments, the carrier may be formulated such that the
composition substantially retains its shape in fluids such as
blood, serum, or water. Such carrier may comprise, for example, a
combination of alginate and chitosan, an acidic alginate (a
combination of alginate and an acid), or other.
Suitable carriers include DBM, including surface demineralized
bone; mineralized bone; nondemineralized cancellous scaffolds;
demineralized cancellous scaffolds; cancellous chips; particulate,
demineralized, guanidine extracted, species-specific (allogenic)
bone; specially treated particulate, protein extracted,
demineralized, xenogenic bone; collagen; synthetic hydroxyapatites;
synthetic calcium phosphate materials; tricalcium phosphate,
sintered hydroxyapatite, settable hydroxyapatite; polylactide
polymers; polyglycolide polymers, polylactide-co-glycolide
copolymers; tyrosine polycarbonate; calcium sulfate; collagen
sheets; settable calcium phosphate; polymeric cements; settable
poly vinyl alcohols, polyurethanes; resorbable polymers; and other
large polymers; liquid settable polymers; and other biocompatible
settable materials. The carrier may further comprise a polyol
(including glycerol or other polyhydroxy compound), a
polysaccharide (including starches), a hydrogel (including
alginate, chitosan, dextran, pluronics, N,O-carboxymethylchitosan
glucosamine (NOCC)), hydrolyzed cellulose, or a polymer (including
polyethylene glycol). In embodiments wherein chitosan is used as a
carrier, the chitosan may be dissolved using known methods
including in water, in mildly acidic aqueous solutions, in acidic
solutions, etc. The carrier may further comprise a hydrogel such as
hyaluronic acid, dextran, Pluronic block copolymers of polyethylene
oxide and polypropylene, and others. Suitable polyhydroxy compounds
include such classes of compounds as acyclic polyhydric alcohols,
non-reducing sugars, sugar alcohols, sugar acids, monosaccharides,
disaccharides, water-soluble or water dispersible oligosaccharides,
polysaccharides and known derivatives of the foregoing. An example
carrier comprises glyceryl monolaurate dissolved in glycerol or a
4:1 to 1:4 weight mixture of glycerol and propylene glycol.
Reference is made to U.S. Pat. No. 5,314,476 for other carriers
including polyhydroxy carriers, to U.S. Pat. No. 6,884,778 for
biocompatible macromere that may be used as carriers, and to U.S.
Patent Publication No. 2003/0152548 for cross-linkable monomers
that may be used as carriers, all herein incorporated by reference.
Settable materials may be used, and they may set up either in situ,
or prior to implantation. In embodiments where alginate salt
(alginate sodium) is used as a settable carrier, the alginate
sodium may be dissolved in water with mild acids. After adding
partially demineralized bone, including surface demineralized bone,
a reaction may occur between acid in alginate solution and minerals
in bone to release calcium ions, which may cross-link alginate to
help set the formulation. Optionally, xenogenic bone powder
carriers also may be treated with proteases such as trypsin.
Xenogenic carriers may be treated with one or more fibril modifying
agents to increase the intraparticle intrusion volume (porosity)
and surface area. Useful agents include solvents such as
dichloromethane, trichloroacetic acid, acetonitrile and acids such
as trifluoroacetic acid and hydrogen fluoride. The choice of
carrier may depend on the desired characteristics of the
composition. In some embodiments, a lubricant, such as water,
glycerol, or polyethylene glycol may be added.
In some embodiments, the osteoinductive composition may comprise
surface demineralized bone particles, demineralized bone matrix,
tissue-derived extract such as collagenous extract, and glycerol.
The osteoinductive composition may be configured to be moldable,
extrudable, or substantially solid. The osteoinductive composition
may be configured to substantially retain its shape in water for a
period of time.
Any suitable shape, size, and porosity of carrier may be used. In
some embodiments, the carrier may be settable and/or injectable.
Such carrier may be, for example, a polymeric cement, a settable
calcium phosphate, a settable poly vinyl alcohol, a polyurethane,
or a liquid settable polymer. Suitable settable calcium phosphates
are disclosed in U.S. Pat. Nos. 5,336,264 and 6,953,594, herein
incorporated by reference. Hydrogel carriers may additionally
impart improved spatial properties, such as handling and packing
properties, to the osteoconductive composition. An injectable
carrier may be desirable where the composition is used with a
covering. Generally, the carrier may have several functions. In
some embodiments, it carries the tissue-derived extract and
partially demineralized bone and allows appropriate release
kinetics. The carrier may also accommodate each step of the
cellular response during bone development, and in some cases
protect the tissue-derived extract from nonspecific proteolysis. In
addition, selected materials must be biocompatible in vivo and
optionally biodegradable. In some uses, the carrier acts as a
temporary scaffold until replaced by new bone. Polylactic acid
(PLA), polyglycolic acid (PGA), and various combinations have
different dissolution rates in vivo. In bone, the dissolution rates
can vary according to whether the composition is placed in cortical
or trabecular bone.
The carrier may comprise a shape-retaining solid made of loosely
adhered particulate material, e.g., with collagen. It may
alternatively comprise a molded, porous solid, a monolithic solid,
or an aggregate of close-packed particles held in place by
surrounding tissue. Masticated muscle or other tissue may also be
used. Large allogenic bone implants may act as a carrier, for
example where their marrow cavities are cleaned and packed with
particles and the osteoinductive factors.
In one embodiment, the osteoinductive composition induces
endochondral bone formation reliably and reproducibly in a
mammalian body. The carrier may comprise particles of porous
materials. The pores may be of a dimension to permit progenitor
cell migration into the carrier and subsequent differentiation and
proliferation. The particle size thus may be within the range of
approximately 70 .mu.m to approximately 850 .mu.m, from 70 .mu.m to
approximately 420 .mu.m, or from approximately 150 .mu.m to
approximately 420 .mu.m. It may be fabricated by close packing
particulate material into a shape spanning the bone defect, or by
otherwise structuring as desired a material that is biocompatible,
and preferably biodegradable in vivo to serve as a "temporary
scaffold" and substratum for recruitment of migratory progenitor
cells, and as a base for their subsequent anchoring and
proliferation. For such embodiments, useful carrier materials
include collagen; homopolymers or copolymers of glycolic acid,
lactic acid, and butyric acid, including derivatives thereof; and
ceramics, such as hydroxyapatite, tricalcium phosphate and other
calcium phosphates. Combinations of these carrier materials also
may be used.
One way to protect small size particles from cellular ingestion
and/or to provide a diffusion barrier is to embed them in a
monolithic bioabsorbable matrix, and then fragment the
particle-containing monolithic matrix into particle sizes greater
than 70 microns, for example, greater than 100 microns, or greater
than 150 microns in their smallest dimension. Suitable matrices for
embedding small partially demineralized particles include
biocompatible polymers and setting calcium phosphate cements.
Generally the particulate partially demineralized bone/polymer
weight ratio will range from about 1:5 to about 1:3. In the case of
calcium phosphate, the partially demineralized bone will be present
up to 75% by weight. Particulation of a monolith can be
accomplished by conventional milling or grinding, or through the
use of cryomilling, or freezing followed by pulverization. In one
embodiment, partially demineralized bone particles are embedded in
a resorbable polymer. In a further embodiment, partially
demineralized bone particles are embedded in one of the setting
calcium phosphates known to the art.
The carrier may comprise a number of materials in combination, some
or all of which may be in the form of fibers and/or particles. The
carrier may comprise calcium phosphates. Driessens et al. "Calcium
phosphate bone cements," Wise, D. L., Ed., Encyclopedic Handbook of
Biomaterials and Bioengineering, Part B, Applications New York:
Marcel Decker; Elliott, Structure and Chemistry of the Apatites and
Other Calcium Phosphates Elsevier, Amsterdam, 1994, each of which
is herein incorporated by reference. Calcium phosphate matrices
include, but are not limited to, dicalcium phosphate dihydrate,
monetite, tricalcium phospate, tetracalcium phosphate,
hydroxyapatite, nanocrystalline hydroxyapatite, poorly crystalline
hydroxyapatite, substituted hydroxyapatite, and calcium deficient
hydroxyapatites.
In one embodiment, the carrier comprises an osteoinductive material
such as a mineralized particulated material, osteoinductive growth
factors, or partially demineralized bone. The mineralized
particulated material may be TCP, hydroxyapatite, mineral recovered
from bone, cancellous chips, cortical chips, surface demineralized
bone, or other material. The osteoinductive material may be
combined with a further carrier such as starch or glycerol.
Accordingly, in some embodiments, the partially demineralized bone
may act as a carrier for the tissue-derived extract.
The osteoinductive composition, comprising partially demineralized
bone and, in some embodiments, tissue-derived extract and carrier,
may be completely insoluble or may be slowly solubilized after
implantation. Following implantation, the composition may resorb or
degrade, remaining substantially intact for at least one to seven
days, or for two or four weeks or longer and often longer than 60
days. The composition may thus be resorbed prior to one week, two
weeks, three weeks, or other, permitting the entry of bone healing
cells.
In various embodiments, the partially demineralized bone may be
bonded together to provide a solid, coherent aggregate through
engagement with particles of binding agent present on the surfaces
of the partially demineralized bone. Reference is made to U.S. Pat.
Nos. 6,696,073, 6,478,825, 6,440,444, and 6,294,187, and to U.S.
Patent Publications Nos. 2006/0216323 and 2005/0251267, all herein
incorporated by reference.
Provide Partially Demineralized Bone in Covering
As shown in block 22 of FIG. 1, in some embodiments the
composition, including the surface-demineralized bone particles,
pressed demineralized bone fibers, tissue derived extract, and/or
carrier, may be provided in a containment covering, such as a
porous mesh, to provide a delivery system. Generally, the covering
may be biocompatible and resorbable.
In some embodiments, surface demineralized bone particles, and
optionally demineralized bone fibers, may be provided in a covering
such that the covering provides a focus or concentration of
biological activity and maintains the surface demineralized bone
particles and demineralized bone fibers in spatial proximity to one
another, possibly to provide a synergistic effect. The covering
further may control availability of the surface demineralized bone
particles and demineralized bone fibers to cells and tissues of a
surgical site over time. In some embodiments, the delivery system
may be used for delivery through a limited opening, such as in
minimally invasive surgery or mini-open access. In some
embodiments, the delivery system may be used to deliver morselized
or particulated materials (the substance provided in the covering)
in pre-measured amounts.
The covering may have a single compartment or may have a plurality
of compartments. Thus, in one embodiment, the covering comprises
first and second compartments. The surface demineralized bone
particles may be provided in the first compartment and the
demineralized bone fibers may be provided in the second
compartment. The second compartment may be adjacent, apart from,
inside, or surrounding the first compartment. In alternative
embodiments, a blend of surface demineralized particles,
demineralized bone fibers, tissue-derived extract, and/or other
materials may be provided in either or both of first compartment
and the second compartment.
In use, the partially demineralized bone particles, and
demineralized bone matrix if provided, may be placed in the
covering prior to implantation of the covering in the body. In
alternative embodiments, the covering may be implanted in the body
and the partially demineralized bone particles, and demineralized
bone matrix if provided, may be placed in the covering
thereafter.
In various embodiments, the covering may comprise a polymer (such
as polyalkylenes (e.g., polyethylenes, polypropylenes, etc.),
polyamides, polyesters, polyurethanes, poly(lactic acid-glycolic
acid), poly(lactic acid), poly(glycolic acid), poly(glaxanone),
poly(orthoesters), poly(pyrolicacid), poly(phosphazenes), L-co-G,
etc.), other bioabsorbable polymer such as Dacron or other known
surgical plastics, a natural biologically derived material such as
collagen, a ceramic (with bone-growth enhancers, hydroxyapatite,
etc.), PEEK (polyether-etherketone), desicated biodegradable
material, metal, composite materials, a biocompatible textile
(e.g., cotton, silk, linen), or other. In one embodiment, the
containment covering is formed as a long tube-like covering and may
be used with minimally invasive techniques.
IX. Form an Implant
The osteoimplant resulting from the partially demineralized bone,
demineralized bone matrix, tissue-derived extract, and/or carrier
may be flowable, have a putty or gel-like consistency, may be
shaped or molded, may be provided as a slurry, may be deformable,
and/or may comprise substantially dry pieces held together in a
covering. The osteoimplant may comprise a monolithic bone or may
comprise an aggregate of smaller bone elements. The osteoimplant
may assume a determined or regular form or configuration such as a
sheet, plate, disk, tunnel, cone, or tube, to name but a few.
Prefabricated geometry may include, but is not limited to, a
crescent apron for single site use, an I-shape to be placed between
teeth for intra-bony defects, a rectangular bib for defects
involving both the buccal and lingual alveolar ridges,
neutralization plates, reconstructive plates, buttress plates,
T-buttress plates, spoon plates, clover leaf plates, condylar
plates, compression plates, bridge plates, or wave plates. Partial
tubular as well as flat plates can be fabricated from the
osteoimplant. Such plates may include such conformations as, e.g.,
concave contoured, bowl shaped, or defect shaped. The osteoimplant
can be machined or shaped by any suitable mechanical shaping means.
Computerized modeling can provide for the intricately-shaped
three-dimensional architecture of an osteoimplant custom-fitted to
the bone repair site with great precision. In embodiments wherein
the osteoimplant is shaped or moldable, the implant may retain
coherence in fluids.
Accordingly, the osteoinductive composition, especially when
comprising as an aggregate of particles, may be subjected to a
configuring step to form an osteoimplant. The configuring step can
be employed using conventional equipment known to those skilled in
the art to produce a wide variety of geometries, e.g., concave or
convex surfaces, stepped surfaces, cylindrical dowels, wedges,
blocks, screws, and the like. A surgically implantable material
fabricated from elongated bone particles that have been
demineralized, which may be shaped as a sheet, and processes for
fabricating shaped materials from demineralized bone particles is
disclosed in U.S. Pat. Nos. 5,507,813 and 6,436,138, respectively,
the contents of which are herein incorporated by reference.
Suitable sheets include those sold under the trade name
Grafton.RTM. DBM Flex, which must be wetted/hydrated prior to use
to be useful for implantation. Such sheets have recently been
reported as effective in seeding human bone marrow stromal cells
(BMSCs), which may be useful in the repair of large bone defects.
Kasten et al., "Comparison of Human Bone Marrow Stromal Cells
Seeded on Calcium-Deficient Hydroxyapatite, Betatricalcium
Phosphate and Demineralized Bone Matrix," Biomaterials,
24(15):2593-603, 2003. Also useful are demineralized bone and other
matrix preparations comprising additives or carriers such as
binders, fillers, plasticizers, wetting agents, surface active
agents, biostatic agents, biocidal agents, and the like. Some
exemplary additives and carriers include polyhydroxy compounds,
polysaccharides, glycosaminoglycan proteins, nucleic acids,
polymers, poloxamers, resins, clays, calcium salts, and/or
derivatives thereof.
In some embodiments, the osteoinductive composition may have
improved spatial properties, such as material handling and packing
properties. Unlike DBM, surface demineralized or mineralized
particles do not generally entangle and hold together.
Tissue-derived extracts having large amounts of collagen type I or
collagen type I residues, for example a collagenous extract, can
impart handling and packing properties to surface demineralized
bone particles. Thus, an osteoinductive composition comprising
surface demineralized bone particles and such tissue-derived
extract generally may have better remodeling properties than
surface demineralized bone alone. The improved remodeling
properties can further be enhanced by a carrier. In some
embodiments, the partially demineralized bone particles may be
forced into close proximity, resulting in better osteoconduction.
Some carriers may be especially suited for providing improved
material handling and packing properties. These include, for
example hydrogels such as chitosan and fast resorbing formulations
of L-co-G. In some embodiments, the osteoinductive composition may
comprise partially or fully demineralized bone particles having an
improved packing efficiency.
X. Formulation
The osteoinductive composition, the delivery vehicle (including
carrier or covering), or the osteoimplant may be formulated for a
particular use. The formulation may be used to alter the physical,
biological, or chemical properties of the composition or the
carrier. A physician would readily be able to determine the
formulation needed for a particular application, taking into
account such factors as the type of injury, the site of injury, the
patient's health, and the risk of infection. In various
embodiments, the osteoinductive composition may comprise, for
example less than approximately 0.5% water, less than approximately
1% water, or less than approximately 5% water.
Osteoinductive compositions or osteoimplants therefore may be
prepared to have selected resorption/loss of osteoinductivity
rates, or even to have different rates in different portions of an
implant. For example, the formulation process may include the
selection of partially demineralized particles of a particular size
or composition, combined with the selection of a particular
stabilizing agent or agents, and the amounts of such agents.
In one example, an osteoimplant may be provided whose
tissue-derived extract comprises osteoinductive factors that are
active in a relatively constant amount over a given period of time.
An osteoimplant comprising factors with longer half-lives can be
prepared using a less biodegradable polymer or a larger amount
(e.g., a thicker coating) of polymeric compound. Alternatively or
additionally, the particle size of the partially demineralized bone
may be important in determining the half-life of the osteoimplant.
In certain embodiments, an osteoinductive composition may include a
mixture of particles, each with a different half-life. Such a
mixture could provide the steady or possible unmasking of
osteoinductive factors over an extended period of time ranging from
days to weeks to months depending on the needs of the injury.
Compositions such as this can be formulated to stimulate bone
growth in a human patient comparable to the bone growth induced by
treatment with 10 .mu.g of rhBMP on a collagen sponge, and
preferably comparable to 100 .mu.g, and most preferably 1-10 mg
rhBMP. When the degradation of the osteoimplant is of concern, it
may be desirable to test the shelf-life of the osteoimplant to
determine shelf-life at, for example, 1, 2, or 3 years. This may be
done by storing the osteoimplant at, for example, room temperature
or, for accelerated testing, 38.degree. C., and periodically
checking the inductivity of the osteoimplant. Reference is made to
PCT/US05/003092, which is hereby incorporated by reference herein.
Implants with enhanced shelf lives may retain more than about 75%
and about 80% of their osteoinductivity after as long as, or longer
than, three years.
Physical properties such as deformability and viscosity of the
carrier may also be chosen depending on the particular clinical
application. The partially demineralized bone may be mixed with
other materials and factors to improve other characteristics of the
implant. For example, the partially demineralized bone may be mixed
with other agents to improve wound healing. These agents may
include drugs, proteins, peptides, polynucleotides, solvents,
chemical compounds, and biological molecules.
Further, the composition may be formulated to be settable and/or
injectable. Thus, for example, the composition may be injectable
through a 10-gauge, a 12-gauge, or an 18-gauge needle.
Accordingly, in some embodiments the composition may be
substantially solid pieces, rubbery, rubbery with chunks, stiff (as
freeze-dried), stiff with chunks, putty, paste, flowable, or
injectable. The term "flowable" in this context applies to
compositions whose consistencies range from those which can be
described as shape-sustaining but readily deformable, e.g., those
which behave like putty, to those which are runny. Specific forms
of flowable bone powder compositions include cakes, pastes, creams
and fillers. Reference is made to U.S. Pat. No. 5,290,558, herein
incorporated by reference in its entirety, for discussion of
flowable materials.
Also as previously discussed, the osteoinductive composition may be
formed into various shapes and configurations, including rods,
strings, sheets, weaves, solids, cones, discs, fibers, and wedges.
Such shapes may result from a monolithic bone piece or an aggregate
of bone particles. In certain embodiments, the shape and size of
the partially demineralized bone affect the time course of
osteoinductivity. For example, in a cone or wedge shape, the
tapered end will result in osteoinductivity shortly after
implantation of the osteoimplant, whereas the thicker end will lead
to osteoinductivity later in the healing process (hours to days to
weeks later). In certain embodiments of osteoimplants comprising an
aggregate of bone particles, the particles have a length of greater
than 2 mm, greater than 1.5 mm, greater than 1 mm, greater than 500
microns, or greater than 200 microns across its widest dimension.
Also, larger particle size will induce bone formation over a longer
time course than smaller particles. Particles of different
characteristics (e.g., composition, size, shape) may be used in the
formation of these different shapes and configurations. For
example, in a sheet of partially demineralized bone, a layer of
long half-life particles may be alternated between layers of
shorter half-life particles. See U.S. Pat. No. 5,899,939, herein
incorporated by reference, for suitable examples. In a weave,
strands composed of short half-life particles may be woven together
with strands of longer half-lives.
In one embodiment, fibrous partially demineralized bone may be
shaped into a matrix form as described in U.S. Pat. No. 5,507,813,
herein incorporated by reference. The shaped partially
demineralized bone may then be embedded within a diffusion barrier
type matrix, such that a portion of the matrix is left exposed free
of the matrix material. Suitable blocking matrices are starch,
phosphatidyl choline, tyrosine polycarbonates, tyrosine
polyarylates, polylactides, polygalactides, or other resorbable
polymers or copolymers. Devices prepared in this way from these
matrices have a combination of immediate and longer lasting
osteoinductive properties and are particularly useful in promoting
bone mass formation in human posterolateral spine fusion
indications.
In another embodiment, carriers having a pre-selected
three-dimensional shape may be prepared by repeated application of
individual layers of partially demineralized bone, for example by
3-D printing as described by U.S. Pat. Nos. 5,490,962, 5,518,680,
and 5,807,437, each incorporated herein by reference. Different
layers may comprise individual stabilized partially demineralized
bone preparations, or alternatively may comprise partially
demineralized bone layers treated with stabilizing agents after
deposition of multiple layers.
In the process of preparing the osteoimplant, the materials may be
produced entirely aseptically or be sterilized to eliminate any
infectious agents such as HIV, hepatitis B, or hepatitis C. The
sterilization may be accomplished using antibiotics, irradiation,
chemical sterilization (e.g., ethylene oxide), or thermal
sterilization. Other methods known in the art of preparing DBM such
as defatting, sonication, and lyophilization may also be used in
preparing a DBM carrier. Since the biological activity of
demineralized bone is known to be detrimentally affected by most
terminal sterilization processes, care must be taken when
sterilizing the inventive compositions.
XI. Optional Additives
Optionally, other additives may be included in the osteoconductive
composition. It will be appreciated that the amount of additive
used will vary depending upon the type of additive, the specific
activity of the particular additive preparation employed, and the
intended use of the composition. The desired amount is readily
determinable by the user.
Any of a variety of medically and/or surgically useful optional
substances can be incorporated in, or associated with, the
osteoinductive factors either before, during, or after preparation
of the osteoinductive composition. Thus, for example when
demineralized bone particles are used to form the material, one or
more of such substances may be introduced into the demineralized
bone particles, for example, by soaking or immersing the bone
particles in a solution or dispersion of the desired
substance(s).
Medically/surgically useful substances that can be readily combined
with the partially demineralized bone include, for example,
collagen, insoluble collagen derivatives, etc., and soluble solids
and/or liquids dissolved therein, e.g., antiviricides, particularly
those effective against HIV and hepatitis; antimicrobials and/or
antibiotics such as erythromycin, bacitracin, neomycin, penicillin,
polymyxin B, tetracyclines, viomycin, chloromycetin and
streptomycins, cefazolin, ampicillin, azactam, tobramycin,
clindamycin and gentamicin, etc.; biocidal/biostatic sugars such as
dextroal, glucose, etc.; amino acids, peptides, vitamins, inorganic
elements, co-factors for protein synthesis; hormones; endocrine
tissue or tissue fragments; synthesizers; enzymes such as
collagenase, peptidases, oxidases, etc.; polymer cell scaffolds
with parenchymal cells; angiogenic drugs and polymeric carriers
containing such drugs; collagen lattices; antigenic agents;
cytoskeletal agents; cartilage fragments, living cells such as
chondrocytes, bone marrow cells, mesenchymal stem cells, natural
extracts, tissue transplants, bone, demineralized bone powder,
autogenous tissues such blood, serum, soft tissue, bone marrow,
etc.; bioadhesives, bone morphogenic proteins (BMPs), angiogenic
factors, transforming growth factor (TGF-beta), insulin-like growth
factor (IGF-1); growth hormones such as somatotropin; bone
digestors; antitumor agents; immuno-suppressants; permeation
enhancers, e.g., fatty acid esters such as laureate, myristate and
stearate monoesters of polyethylene glycol, enamine derivatives,
alpha-keto aldehydes, etc.; and, nucleic acids. The amounts of such
optionally added substances can vary widely with optimum levels
being readily determined in a specific case by routine
experimentation.
Bone regeneration involves a multitude of cells (e.g. cartilage,
fibroblasts, endothelial, etc.) besides osteoblasts. Stem cells may
be combined with the partially demineralized bone. Accordingly, the
osteoinductive composition may be used to deliver stem cells, which
offers the potential to give rise to different types of cells in
the bone repair process
In certain embodiments, the additive is adsorbed to or otherwise
associated with the osteoinductive composition. The additive may be
associated with the osteoinductive composition through specific or
non-specific interactions, or covalent or noncovalent interactions.
Examples of specific interactions include those between a ligand
and a receptor, an epitope and an antibody, etc. Examples of
nonspecific interactions include hydrophobic interactions,
electrostatic interactions, magnetic interactions, dipole
interactions, van der Waals interactions, hydrogen bonding, etc. In
certain embodiments, the additive is attached to the osteoinductive
composition, for example, to the carrier, using a linker so that
the additive is free to associate with its receptor or site of
action in vivo. In other embodiments the additive is either
covalently or non-covalently attached to the carrier. In certain
embodiments, the additive may be attached to a chemical compound
such as a peptide that is recognized by the carrier. In another
embodiment, the additive is attached to an antibody, or fragment
thereof, that recognizes an epitope found within the carrier. In
certain embodiments at least additives are attached to the
osteoimplant. In other embodiments at least three additives are
attached to the osteoinductive composition. An additive may be
provided within the osteoinductive composition in a sustained
release format. For example, the additive may be encapsulated
within biodegradable nanospheres, microspheres, etc.
It will be understood by those skilled in the art that the lists of
optional substances herewith included are not intended to be
exhaustive and that other materials may be admixed with
bone-derived elements within the practice of the present
invention.
In one embodiment, the osteoconductive composition further
comprises a cell such as an osteogenic cell or a stem cell. In
various embodiments, the additive may comprise radiopaque
substances, angiogenesis promoting materials, bioactive agents,
osteoinducing agents, or other. Reference is made to U.S. patent
application Ser. Nos. 11/555,606 and 11/555,608 for specific
discussion of possible additives.
XII. Assessment of Osteogenic Activity
Any suitable manner for assessing osteogenic activity may be used.
Generally, the more closely the manner of assessing
osteoinductivity correlates with the anticipated use of the
osteoinductive composition, the more predictive the results will be
of how the osteoinductive composition will perform in a human.
Thus, for example, a sheep vertebral model may be used to assess
osteogenic activity of the osteoinductive composition.
In various embodiments, the osteoinductive composition may have an
inductivity exceeding that of between 2 and 20 volumes of
mineralized bone that is prepared into demineralized bone. For
example, the osteoinductive composition may have an inductivity
exceeding that of approximately five volumes of mineralized bone
that is prepared into demineralized bone. In some embodiments, one
gram of the osteoinductive composition may have inductivity
exceeding that of demineralized bone prepared from five grams of
mineralized allograft bone.
Induction of bone formation can be determined by a histological
evaluation showing the de novo formation of bone with accompanying
osteoblasts, osteoclasts, and osteoid matrix. For example,
osteoinductive activity of an osteoinductive factor can be
demonstrated by a test using a substrate onto which material to be
tested is deposited. The substrate with deposited material is
implanted subcutaneously in a test animal. The implant is
subsequently removed and examined microscopically for the presence
of bone formation including the presence of osteoblasts,
osteoclasts, and osteoid matrix. A suitable procedure for assessing
osteoinductive activity is illustrated in Example 5 of U.S. Pat.
No. 5,290,763, herein incorporated by reference. Although there is
no generally accepted scale of evaluating the degree of osteogenic
activity, certain factors are widely recognized as indicating bone
formation. Such factors are referenced in the scale of 0-8 which is
provided in Table 3 of example 1 of U.S. Pat. No. 5,563,124, herein
incorporated by reference. The 0-4 portion of this scale
corresponds to the scoring system described in U.S. Pat. No.
5,290,763, which is limited to scores of 0-4. The remaining portion
of the scale, scores 5-8, references additional levels of
maturation of bone formation. The expanded scale also includes
consideration of resorption of collagen, a factor which is not
described in the '763 patent. Osteoinductivity may be assessed in
tissue culture, e.g. as the ability to induce an osteogenic
phenotype in culture cells (primary, secondary, cell lines, or
explants). Cell culture assays measure the ability of a matrix to
cause one or more features indicative of differentiation along an
osteoblastic or chondrocytic lineage. The feature(s) can be an
expression of a marker characteristic of differentiation along an
osteoblastic or chondrocytic lineage, e.g. a marker that is
normally expressed by osteoblast precursors, osteoblasts,
chondrocytes, or precursors of chondrocytes. One suitable marker is
alkaline phosphatase. Reference is made to U.S. patent application
Ser. No. 11/683,938, herein incorporated by reference, for
discussion of alternative in vitro assay methods.
In studies, a typical amount of DBM for implantation is 20 mg in a
mouse and 40 mg in a rat. Significant increases in the growth
factor dose, for example, 150.times. dose (or 150 times the growth
factor found in normal DBM), lead to significantly more and
potentially faster bone growth with larger volume bone growth, more
dense bone growth, larger nodules of bone growth, higher x-ray
density, and, generally, a higher osteoinductive score. Associated
with this increase in osteoinductivity can be a cortical shell
surrounding the nodule and some level of vascularization in the
nodule. However, the ability to quantitatively measure is generally
limited by the method used, and generally measured increases in
osteoinductive activity are not linear with the increase in dosage.
Thus, if 20 mg of DBM gives an osteoinductive activity of 1, 100
times the growth factor dose (2000 mg of DBM growth factors) does
not give an osteoinductive activity of 100. Instead, it may result
in an osteoinductive activity of about 20. A limitation of
measurement using osteoinductive scores is that, in some
situations, the system's ability to respond may be saturated. Thus,
for example, if the score ranges only from 1 to 4, two samples may
have the same score (4) but may not, in fact, be comparable. This
is particularly the case when the bone resulting from one method or
implant is qualitatively better than the bone resulting from
another method or implant. That is, both methods or implants may
result in an osteoinductive score of 4 but one may result in
qualitatively better bone than the other. Thus, in some situations
it may be desirable to test speed of growth, density, presence of
cortical bone, shelling, and/or other factors showing an increase
over normal demineralized bone matrix. Further, in addition to, or
in lieu of, testing at 28 days, it may be desirable to test
inductivity at 21 days Generally, inductivity may be measured
histomorphometrically by methods known in art.
Further, delivering 100 times the growth factor dose may be
challenging. In filling a bone defect, only as much filler may be
used as there is bone void space.
XIII. Examples
The examples may refer to particles, particles formed into a putty,
particles formed into a gel, or other. It is to be understood that
the examples are illustrative only and are not intended to be
limiting. Thus, each example may be modified to provide
compositions having differing consistencies such as flowable,
injectable, rubbery, flexible, stiff, or other.
Example 1
Surface Demineralized Heat Treated Particles
In one example, bone was cleaned of soft tissue and ground to
powder ranging from 2.8 mm to 4 mm. The particles were extracted
with 1:1 chloroform-methanol for 6 hours. The solvent was then
decanted and the excess allowed to evaporate under a fume hood
overnight. The particles were then vacuum dried overnight.
The particles were surface demineralized for 75 minutes in 0.6 N
HCl and then washed with distilled water until the pH of the wash
exceeded 3.0. The resulting surface demineralized particles were
then incubated with agitation in 100 mM phosphate buffer, pH 7.4,
containing 6.0 mM NEM and 2.0 mM sodium azide for 72 hours at
37.degree. C.
The resulting particles were washed two times for 15 minutes in
water at room temperature. The particles were lyophilized and
implanted in a sheep femoral defect; the results were examined by
micro-CT analysis 4 weeks and 13 weeks post-implantation.
FIG. 5 illustrates the 13 week results of autograft and of surface
demineralized heat treated particles.
Example 2
Surface Demineralized Heat Treated Particles
The particles are prepared as described in Example 1 excepting
incubation in phosphate buffer.
Example 3a
Smaller Surface Demineralized Heat Treated Particles
Particles were ground to a size ranging from 1 mm to 2.8 mm and
demineralized in 0.6N HCl for 60 minutes prior to heat treatment as
described in Example 1.
Example 3b
Smaller Surface Demineralized Heat Treated Particles
Particles were ground to a size ranging from 0.5 mm to 1.0 mm and
demineralized in 0.6N HCl for 10 minutes prior to heat treatment as
described in Example 1.
Example 3c
Smaller Surface Demineralized Heat Treated Particles
Particles were ground to a size ranging from 0.1 mm to 0.5 mm and
demineralized in 0.6N HCl for 7 minutes prior to heat treatment as
described in Example 1.
Example 4a
Various Degrees of Demineralization
Particles are ground to a size ranging from 1.0 mm to 2.8 mm and
demineralized for 15 minutes prior to heat treatment as described
in Example 1.
Example 4b
Various Degrees of Demineralization
Particles are ground to a size ranging from 1.0 mm to 2.8 mm and
demineralized for 30 minutes prior to heat treatment as described
in Example 1.
Example 4c
Various Degrees of Demineralization
Particles are ground to a size ranging from 1.0 mm to 2.8 mm and
demineralized for 120 minutes prior to heat treatment as described
in Example 1.
Example 4d
Various Degrees of Demineralization
Particles are ground to a size ranging from 1.0 mm to 2.8 mm and
demineralized for 240 minutes prior to heat treatment as described
in Example 1.
Example 4e
Various Degrees of Demineralization
Particles are ground to a size ranging from 1.0 mm to 2.8 mm and
demineralized for 480 minutes prior to heat treatment as described
in Example 1.
Example 4f
Various Degrees of Demineralization
Particles are ground to a size ranging from 1.0 mm to 2.8 mm and
fully demineralized prior to heat treatment as described in Example
1.
Example 4g
Various Degrees of Demineralization
Particles are ground to a size ranging from 2.8 mm to 4.0 mm and
demineralized for 15 minutes prior to heat treatment as described
in Example 1.
Example 4h
Various Degrees of Demineralization
Particles are ground to a size ranging from 2.8 mm to 4.0 mm and
demineralized for 240 minutes prior to heat treatment as described
in Example 1.
Example 4i
Various Degrees of Demineralization
Particles are ground to a size ranging from 2.8 mm to 4.0 mm and
demineralized for 480 minutes prior to heat treatment as described
in Example 1.
Example 4j
Various Degrees of Demineralization
Particles are ground to a size ranging from 2.8 mm to 4.0 mm and
fully demineralized prior to heat treatment as described in Example
1.
Example 4k
Various Degrees of Demineralization
Particles are treated as described in Example 1 and above excepting
incubation in phosphate buffer.
Example 5a
Mixing of Surface Demineralized Particles with DBM Fiber
Particles are made as in Example 1 and mixed with demineralized
bone fibers in a ratio of 3 volumes of surface demineralized
particles to 1 volume of DBM fiber.
Example 5b
Mixing of Surface Demineralized Particles with DBM Fiber
Particles are made as in Example 1 and mixed with demineralized
bone fibers in a ratio of 1 volume of surface demineralized
particles to 1 volume of DBM fiber.
Example 5c
Mixing of Surface Demineralized Particles with DBM Fiber
Particles are made as in Example 1 and mixed with demineralized
bone fibers in a ratio of 2 volume of surface demineralized
particles to 1 volume of DBM fiber.
Example 6a
Combining Surface Demineralized Heat Treated Particles with DBM
Extracts
Particles made as in Example 1 are mixed with protein extracted
from an equal volume of demineralized bone matrix with 4 M
Guanidine HCl. The extracted proteins are added to the surface
demineralized particles and the suspension is dialyzed against
water until the guanidine is effectively removed. The preparation
is then lyophilized.
Example 6b
Combining Surface Demineralized Heat Treated Particles with DBM
Extracts
Particles made as in Example 1 are mixed with protein extracted
from twice the volume of demineralized bone matrix with 4 M
Guanidine HCl. The extracted proteins are added to the surface
demineralized particles and the suspension is dialyzed against
water until the guanidine is effectively removed. The preparation
is then lyophilized.
Example 6c
Combining Surface Demineralized Heat Treated Particles with DBM
Extracts
Particles made as in Example 1 are mixed with protein extracted
from five times the volume of demineralized bone matrix with 4 M
Guanidine HCl. The extracted proteins are added to the surface
demineralized particles and the suspension is dialyzed against
water until the guanidine is effectively removed. The preparation
is then lyophilized.
Example 6d
Combining Surface Demineralized Heat Treated Particles with DBM
Extracts
Particles made as in Example 1 are mixed with protein extracted
from ten times the volume of demineralized bone matrix with 4 M
Guanidine HCl. The extracted proteins are added to the surface
demineralized particles and the suspension is dialyzed against
water until the guanidine is effectively removed. The preparation
is then lyophilized.
Example 7
Organic Precipitation of Proteins
Materials are prepared as in Example 6 excepting precipitation of
proteins onto surface demineralized bone with a volume of 1:1
acetone/ethanol equal to the volume of guanidine HCl.
Example 8
Combining Surface Demineralized Heat Treated Particles with
Demineralized Bone Fibers and Protein Extracts
Mixtures of surface demineralized particles and demineralized bone
matrix fibers described in Example 5 are combined with extracts as
described in Examples 6 and 7.
Example 9
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with
glycerol.
Example 9b
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with a
polylactide polymer.
Example 9c
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with a
polyglycolide polymer.
Example 9d
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with a
polylactide-co-glycolide copolymer.
Example 9e
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with a
starch.
Example 9f
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with
an alginate.
Example 9g
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with
chitosan.
Example 9h
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with a
pluronic.
Example 9i
Combining Surface Demineralized Heat Treated Particles with a
Carrier
Compositions as prepared by any of Examples 1-8 are combined with
hyaluronic acid.
Example 10
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles, DBM fibers and DBM powder (106-500
.mu.m). DBM powder is extracted with 4M guanidine HCl. The
guanidine hydrochloride extract is dialyzed against water and the
supernatant and precipitate are separated via centrifugation. The
collagenous supernatant is lyophilized to obtain dry collagen
residue. 10 grams of dry surface demineralized bone are combined
with 3.85 grams of dry DBM fiber and 0.85 grams dry collagen
residue. The material is mixed in the presence of 20 ml water. The
final mixture is injected into a mold, lyophilized to form a
matrix.
Example 11
Composition is formed as in Example 10 but omitting the
centrifugation of the extract and separation of supernatant from
precipitate.
Example 12
Composition is formed as in Example 10 excepting that mixing in the
final step is carried out in a solution of glycerol and water in a
volume ratio of 45:55.
Example 13
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles and DBM fibers. 10 grams of dry
surface demineralized bone are combined with 3.85 grams of dry DBM
fiber and 0.15 grams of chitosan. Prior to mixing the chitosan is
dissolved in 5 ml of 2% acetic acid. The materials are mixed in the
presence of 15 ml water. The final mixture is injected into a mold,
lyophilized to form a matrix. The matrix is then treated with 5%
sodium citrate for 1 hour, washed and lyophilized.
Example 14
Composition is prepared as in example 13 excepting the use of 15 ml
45:55 glycerol-water in place of 15 ml water and excluding
treatment with sodium citrate.
Example 15
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles and DBM powder (106-500 .mu.m). DBM
powder is extracted with 4M guanidine HCl. The guanidine
hydrochloride extract is dialyzed against water and the supernatant
and precipitate are separated via centrifugation. The collagenous
supernatant is lyophilized to obtain dry collagen residue. 10 g
surface demineralized particles are wetted in 40 ml DI water and
then pressed at 4000 psi. Pressed surface demineralized particles
are soaked in a mixture of glycerol/water (45/55) for 1 hour and
then filtered to get around 23 grams of glycerated material. The
glycerated surface demineralized particles are further combined
with 1.1 grams of collagen residue in 5 ml water. The final mixture
is injected into a mold and lyophilized to obtain a matrix.
Example 16
Composition prepared as in Example 14 but omitting the
centrifugation step and separation of supernatant from
precipitate.
Example 17
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles and DBM fibers. 10 grams of dry
surface demineralized bone are combined with 3.85 grams of dry DBM
fiber and 0.10 grams of human or bovine derived antelocollagen.
Prior to mixing the collagen is suspended in 10 ml of 2% lactic
acid. The materials are mixed in the presence of 10 ml water. The
final mixture is injected into a mold, lyophilized to form a
matrix.
Example 18
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles and DBM fibers. 10 grams of dry
surface demineralized bone are combined with 3.85 grams of dry DBM
fiber and 0.50 grams of polymer. Polymers can be naturally derived
or synthetic such as alginate, cellulose, gelatin, poly(lactic
acid), poly(lactic-co-glycolic acid), poly(caprolactone),
poly(lactide-co-caprolactone), poly(carbonate), Pluronic F127 etc.
Prior to mixing the polymers are dissolved in a biocompatible
solvent. The components are mixed and injected into a mold,
lyophilized to form a matrix.
Example 19
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles, DBM fibers and DBM powder (106-500
.mu.m). DBM powder is extracted with 4M guanidine HCl. The
guanidine hydrochloride extract is dialyzed against water and the
supernatant and precipitate are separated via centrifugation. The
collagenous supernatant is lyophilized to obtain dry collagen
residue. 10 grams of dry surface demineralized bone are combined
with 3.85 grams of dry DBM fiber and 0.85 grams dry collagen
residue. The material is mixed in the presence of 20 ml of water
and loaded into a syringe. Any excess water is extruded.
Example 20
Composition is prepared as in example 19 but omitting the
centrifugation of the extract and separation of supernatant from
precipitate.
Example 21
Composition is prepared as in Example 19 excepting that mixing in
the final step is carried out in a solution of glycerol and water
in a volume ratio of 45:55.
Example 22
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles and DBM fibers. 10 grams of dry
surface demineralized bone are combined with 3.85 grams of dry DBM
fiber and 0.15 grams of chitosan. Prior to mixing the chitosan is
dissolved in 5 ml of 2% acetic acid. The materials are mixed in the
presence of 15 ml water. The material is loaded into a syringe to
obtain an extrudable formulation.
Example 23
Composition is prepared as in Example 22 excepting the use of 15 ml
45:55 glycerol-water in place of 15 ml water.
Example 24
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles, DBM fibers. 10 grams of dry
surface demineralized bone are combined with 3.85 grams of dry DBM
fiber and 8.3 grams of hydrated starch. The material is loaded into
a syringe to obtain an extrudable formulation.
Example 25
Sheep cortical bone is processed to different components: 1-2.8 mm
surface demineralized particles and DBM fibers. 10 grams of dry
surface demineralized bone are combined with 3.85 grams of dry DBM
fiber and 0.10 grams of human or bovine derived predominantly type
I collagen. Prior to mixing the collagen is suspended in 10 ml of
2% lactic acid. The materials are mixed in the presence of 10 ml
water. The material is loaded into a syringe to obtain an
extrudable formulation.
XIV. Assessment of Bone Particles
It may be useful to assess characteristics of the bone particles at
times before, during, or after the methods provided herein.
Assessment of Neutral Protease Activity
It may be useful to assess endogenous protease activity in the bone
particles. For example, in the method shown in FIG. 2, the neutral
protease activity of mineralized bone is high but is reduced upon
demineralization. Accordingly, the surface of the particles after
demineralization has a lower protease activity than prior to
demineralization. The lower protease activity allows maintenance of
osteoinductive activity at the particle surface. Any suitable
method of assessing protease activity may be used.
In one embodiment, the following procedure was used to assess
endogenous protease activity of the bone particles. A Pierce
QuantiCleave Protease Activity Kit was used. In the embodiment
herein described, a modified casein substrate was used. The kit
identifies exposed N-terminal amines of peptides released from the
casein precursor.
All samples were processed using sterile technique. Microfuge tubes
and pipette tips were autoclaved.
Generation of Finely Powdered Sterile DBM and Nondemineralized
Bone
A. 1 gram of human DBM was prepared and finely powdered in a Spec
Freezer Mill using the following protocol:
5 min pre-cool in LN2. (T3)
3.times.2 min cycles (T1)
1 min interim cooling. (T2)
B. 1 gram of powdered nondemineralized bone (mixed batches),
cleaned and sonicated in ethanol, was treated as above.
Preparation of Assay Solution
PBS was used as the Assay Buffer instead of 50 mM Borate Buffer.
This comprised adding 5 ml of PBS to 3 vials (10 mg) of
Succinylated casein, letting stand for 5 min and gently swirling to
dissolve the protein. The contents of three vials were sterile
filtered into a single 15 ml SterileTube. This is known as the
sterile succinylated casein solution. The volume of the sterile
succinylated casein solution was adjusted to 15 ml using sterile
PBS.
300 .mu.l of succinylated casein solution was added to each of five
tubes from each group.
As blanks, 300 .mu.l of phosphate buffered saline, pH 7.4,
containing 0.9 mM CaCl.sub.2, 0.2 H.sub.2O and 0.5 mM MgCl.sub.2
was added to five tubes from each group.
All tubes were vortexed for 20 seconds and then placed on ice for
15 minutes. The tubes were centrifuged at 12,000 rpm for 5 minutes
and the vortexing and centrifugation steps were repeated.
Protease Assay
TPCK trypsin stock solution was prepared by adding 5 mg TPCK
trypsin (included with Kit) to 2.5 ml of PBS. The solution was
sterile filtered into a sterile 15 ml tube and the volume was
raised to 10 ml.
The stock solution was serially diluted in a sterile hood by adding
1 ml of stock to 9 mo of PBS, vortexing, and continuing the 10 fold
dilution series for a total of 9 standards ranging from
5.0.times.10.sup.-1 mg/ml to 5.0.times.10.sup.-9 mg/ml.
All samples received an additional 200 ul Casein/or 200 ul PBS.
The Standards
100 ml of succinylated casein solution was added to each of 21
sterile microfuge tubes.
The following tubes were prepared and processed as described:
TABLE-US-00001 1. 0.0 ng/ml trypsin To three of the tubes add 50 ul
of sterile PBS. 2. 0.005 ng/ml trypsin To three of the tubes add 50
ul 5.0 .times. 10.sup.-9 mg/ml trypsin 3. 0.05 ng/ml trypsin To
three of the tubes add 50 ul 5.0 .times. 10.sup.-8 mg/ml trypsin.
4. 0.5 ng/ml trypsin To three of the tubes add 50 ul 5.0 .times.
10.sup.-7 mg/ml trypsin. 5. 5.0 ng/ml trypsin To three of the tubes
add 50 ul 5.0 .times. 10.sup.-6 mg/ml trypsin. 6. 50.0 ng/ml
trypsin To three of the tubes add 50 ul 5.0 .times. 10.sup.-5 mg/ml
trypsin 7. 500.0 ng/ml trypsin To three of the tubes add 50 ul 5.0
.times. 10.sup.-4 mg/ml trypsin 8. 5000 ng/ml trypsin To three of
the tubes add 50 ul 5.0 .times. 10.sup.-3 mg/ml trypsin 9. 50,000
ng/ml trypsin To three of the tubes add 50 ul 5.0 .times. 10.sup.-2
mg/ml trypsin 10. 500,000 ng/ml trypsin To three of the tubes add
50 ul 5.0 .times. 10.sup.-1 mg/ml trypsin
1-9--Received 9 mls PBS 10--Received 10 mls of PBS
For each standard or sample, the following was done.
Process repeated using sterile PBS in place of succinylated casein.
These tubes served as blanks for the standards.
Incubated for 24 hrs at 40.degree. C. in a shaking water bath.
Color Development
At the end of the 120 hrs period, samples were vortexed and
centrifuged at 13,000 rpm for 10 min. 150 .mu.l of supernatant was
removed from each sample and transferred to a 96 well ELISA
plate.
TNBSA working solution was prepared by adding 100 ul of stock TNBSA
solution to 14.9 ml PBS.
In well A1 place 200 .mu.l of water was placed as the path length
plate blank.
50 .mu.l of TNBSA working solution was added to all other
wells.
Incubated for 20 min at room temperature.
Measured absorbance at 405 nm.
Subtracted the average absorbance of each sample group from the
corresponding blank.
Assessment of Depth of Demineralization
It may further be useful to assess the depth of demineralization of
surface demineralized particles. Any suitable method, including
measurement by x-ray, by contact x-ray, by contact microradiograph,
by stain, by embedding in polymer, be microscopic study, or other
may be used.
In one method, the bone particle is placed in 3% basic fuchsin in
order to stain the demineralized surface. The bone particle is
photographed, acquired with Adobe Photoshop 5.0, and analyzed with
Image-Pro Plus 3.1. The actual depth of demineralization is
calculated by measuring the length (pixels) of the stained
demineralized area at several locations (D.sub.p and D.sub.r) for
each time point. The pixel measurements are averaged and converted
to millimeters.
XV. Uses
Therapeutic Uses
The osteoinductive composition or osteoimplant is intended to be
applied at a bone repair site, for example, a site resulting from
injury, defect brought about during the course of surgery,
infection, malignancy, or developmental malformation. The
osteoinductive composition may be used for treatment of metabolic
bone disease, bone healing, cartilage repair, spinal disc repair,
tendon repair, repair of a defect created by disease or surgery,
dural repair and may be further used in a wide variety of
orthopedic, periodontal, neurosurgical, and oral and maxillofacial
surgical procedures. The osteoinductive composition or osteoimplant
may further be used in veterinary applications.
At the time just prior to when the osteoinductive composition or
osteoimplant is to be placed in a defect site, optional materials,
e.g., autograft bone marrow aspirate, autograft bone, preparations
of selected autograft cells, autograft cells containing genes
encoding bone promoting action, etc., can be combined with the
osteoimplant. The osteoimplant can be implanted at the bone repair
site, if desired, using any suitable affixation means, e.g.,
sutures, staples, bioadhesives, screws, pins, rivets, other
fasteners and the like or it may be retained in place by the
closing of the soft tissues around it.
The osteoinductive compositions may also be used as drug delivery
devices. In certain embodiments, association with the
osteoinductive compositions increases the half-life of the relevant
biologically active agent(s). In some embodiments, the drug
delivery devices may be used to deliver osteoinductive growth
factors. Other preferred agents to be delivered include factors or
agents that promote wound healing. However, the osteoinductive
compositions may alternatively or additionally be used to deliver
other pharmaceutical agents including antibiotics, anti-neoplastic
agents, growth factors, hematopoietic factors, nutrients, an other
bioactive agents described above. The amount of the bioactive agent
included with the DBM composition can vary widely and will depend
on such factors as the agent being delivered, the site of
administration, and the patient's physiological condition. The
optimum levels is determined in a specific case based upon the
intended use of the implant.
Non-Therapeutic Uses
In addition to therapeutic uses involving implantation into a
subject, the osteoinductive composition has a number of other uses.
For example, it can be used to generate or culture cell lines,
tissues, or organs having osteogenic or chondrogenic properties. In
particular, cells can be removed from a donor and cultured in the
presence of an osteoinductive composition. The invention includes
such cells as well as tissues and organs derived therefrom. The
cells, tissues, or organs may be implanted into the original donor
after a period of culture in vitro or may be implanted into a
different subject.
XVI. Conclusion
In certain embodiments, the osteoinductive compositions and
associated osteoimplants produce bone or cartilage in an animal
model and/or in human patients with similar timing and at a level
at least 10%, 20%, 35%, 50%, 100%, 200%, 300%, or 400% or greater
osteogenic, osteoinductive or chondrogenic activity than a
corollary carrier that has not been exposed to a treatment or
condition as described herein. One skilled in the art will
appreciate that these values may vary depending on the type of test
used to measure the osteoinductivity or osteogenic or chondrogenic
activity described above. The test results may fall within the
range of 10% to 35%, 35% to 50%, 50% to 100%, 100% to 200%, and
200% to 400%. In certain embodiments, when an osteoimplant is
implanted into a bone defect site, the osteoimplant has an
osteoinductivity score of at least 1, 2, 3, or 4 in an animal model
and/or in humans.
Although the invention has been described with reference to
specific embodiments, persons skilled in the art will recognize
that changes may be made in form and detail without departing from
the spirit and scope of the invention.
* * * * *